Internalizing Anti-CD74 Antibodies and Methods of Use

ABSTRACT

The present invention provides humanized, chimeric and human anti-CD74 antibodies, CD74 antibody fusion proteins, immunoconjugates, vaccines and bispecific that bind to CD74, the major histocompatibility complex (MHC) class-II invariant chain, Ii, which is useful for the treatment and diagnosis of B-cell disorders, such as B-cell malignancies, other malignancies in which the cells are reactive with CD74, and autoimmune diseases, and methods of treatment and diagnosis.

This application is a divisional of U.S. Ser. No. 12/553,566 (Publ. No.20100015048), filed Sep. 3, 2009, which is a divisional of U.S. Ser. No.11/754,902 (Publ. No. 20080138333), filed May 29, 2007, which is adivisional of U.S. Ser. No. 10/377,122 (now issued U.S. Pat. No.7,312,318), filed Mar. 3, 2003, which claimed the benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 60/360,259, filedMar. 1, 2002, and is a continuation-in-part of U.S. Ser. No. 10/350,096(Publ. No. 20030133930), filed Jan. 24, 2003, which was a continuationof U.S. Ser. No. 09/590,284 (now issued U.S. Pat. No. 7,074,403), filedJun. 9, 2000, which claimed the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 60/138,284, filed Jun. 9, 1999, each ofwhich is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to humanized, chimeric and human anti-CD74antibodies or fragments thereof or antibody fusion proteins comprisingat least one anti-CD74 antibody, particularly monoclonal antibodies(mAbs), therapeutic and diagnostic conjugates of humanized, chimeric andhuman anti-CD74 mAbs or fragments thereof, and methods of treating anddiagnosing B cell lymphomas and leukemias, malignancies other thanlymphomas and leukemias in which the cells are positive for the CD74antigen and various autoimmune and immune dysregulation diseases usingthese humanized, chimeric and human anti-CD74 mAbs or fragments thereof.The present invention relates to multivalent and/or multispecificanti-CD74 mAbs or fragments thereof comprising at least one arm of ananti-CD74 mAb or fragment thereof and at least one arm of themultispecific mAb to a noxious substance, such as a pathogenic organism,such as a cancer cell, a parasite or an infectious agent. The presentinvention further relates to an anti-CD74 mAb or fragment thereofconjugated to an antigenic peptide. The humanized, chimeric and humananti-CD74 mAbs, fragments thereof, and conjugates thereof may beadministered alone or as part of a multimodal therapeutic regimen. Thepresent invention relates to DNA sequences encoding humanized, chimericand human anti-CD74 antibodies, and multivalent and/or multispecificanti-CD74 mAbs and fragments thereof, and therapeutic, diagnostic andantigenic conjugates thereof, vectors and host cells containing the DNAsequences, and methods of making the humanized, chimeric and humananti-CD74 antibodies.

2. Background

One of the major goals of immunotherapy is to harness a patient's immunesystem against tumor cells or infectious organisms. With regard tocancer therapy, the object is to direct the patient's immune systemagainst tumor cells. Non-Hodgkins lymphoma (NHL), multiple myeloma, andchronic and acute lymphocytic leukemia are B-cell malignancies thatremain important contributors to cancer mortality. The response of thesemalignancies to various forms of treatment is mixed.

Induction of a T-Iymphocyte response is a critical initial step in ahost's immune response. Activation of T cells results in T cellproliferation, cytokine production by T cells and generation of Tcell-mediated effector functions. T-cell activation requires anantigen-specific signal, often called a primary activation signal, whichresults from stimulation of a clonally-distributed T cell receptor (TcR)present on the surface of the T cell. This antigen-specific signal isusually in the form of an antigenic peptide bound either to a majorhistocompatibility complex (MHC) class I protein or an MHC class IIprotein present on the surface of an antigen-presenting cell (APC). TheMHC molecules in humans are designated as HLA (human leukocyte antigen)molecules.

Class-II molecules are found on a limited number of cell types,primarily B cells, monocytes/macrophages and dendritic cells, and, inmost cases, present peptides derived from proteins taken up from theextracellular environment. MHC class-II are charged in cellularcompartments which communicate with the extracellular environment. Inhumans the MHC-II molecules comprise the HLA-DR, HLA-DQ and HLA-DPmolecules, which occur in various genetically coded alleles. Thus, e.g.,bacterial antigens from the extracellular environment can be taken upand be presented after intracellular processing in theantigen-presenting cells on their cell surface. CD4+T cells recognizepeptides associated with class-II molecules.

The use of targeting monoclonal antibodies conjugated to radionuclidesor other cytotoxic agents offers the possibility of delivering suchagents directly to the tumor site, thereby limiting the exposure ofnormal tissues to toxic agents (Goldenberg, Semin. Nucl. Med., 19: 332(1989)). In recent years, the potential of antibody-based therapy andits accuracy in the localization of tumor-associated antigens have beendemonstrated both in the laboratory and clinical studies (see, e.g.,Thorpe, TIBTECH, 11: 42 (1993); Goldenberg, Scientific American, Science& Medicine, 1: 64 (1994); Baldwin et al., U.S. Pat. Nos. 4,925,922 and4,916,213; Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095; Irieet al., U.S. Pat. No. 5,196,337; Hellstrom et al., U.S. Pat. Nos.5,134,075 and 5,171,665). In general, the use of radio-labeledantibodies or antibody fragments against tumor-associated markers forlocalization of tumors has been more successful than for therapy, inpart because antibody uptake by the tumor is generally low, ranging fromonly 0.01% to 0.001% of the total dose injected (Vaughan et al., Brit.J. Radiol., 60: 567 (1987)). Increasing the concentration of theradiolabel to increase the dosage to the tumor is counterproductive,generally, as this also increases exposure of healthy tissue toradioactivity.

Murine LL1 (mLL1 or murine anti-CD74 antibody) is a specific monoclonalantibody (mAb) reactive with CD74, the HLA Class-II-Iike antigen, i.e.,the invariant chain (Ii determinant) on the surface of B-lymphocytes,monocytes and histiocytes, human B-lymphoma cell lines, melanomas,T-cell lymphomas and a variety of other tumor cell types (Hansen et al.,Biochem. J. 320:293 (1996)). Cell surface-bound LL1 is rapidlyinternalized to the lysosomal compartment and quickly catabolized, muchfaster than other mAbs, such as anti-CD 19 and anti-CD22. Id. Thisinherent property of LL1 overcomes some of the aforementioneddifficulties with immunotherapy.

Murine LL1 was developed by fusion of mouse myeloma cells withsplenocytes from BALB/c mice immunized with preparations from the RajiB-lymphoma cell line (called EPB-1 in Pawlak-Byczkowska et al., Can.Res., 49: 4568 (1989)). The clinical use of mLL1, just as with mostother promising murine antibodies, has been limited by the developmentin humans of a human anti-mouse antibody (HAMA) response. A HAMAresponse is generally not observed following injection of mLL1 Fab′, asevidenced in a bone marrow imaging study using a mLL1 Fab′ labeled with^(99m)Tc. Juweid et. al., Nuc. Med. Camm. 18: 142-148 (1997). However,in some therapeutic and diagnostic uses, a full-length anti-CD74 mAb maybe preferred. This use of the full-length anti-CD74 mAb can limit thediagnostic and therapeutic usefulness of such antibodies and antibodyconjugates, not only because of the potential anaphylactic problem, butalso as a major portion of the circulating conjugate may be complexed toand sequestered by the circulating anti-mouse antibodies. Although theuse of antibody fragments of mLL1 may circumvent the problems ofimmunogenicity, there are circumstances in which whole IgG is moredesirable and the induction of cellular immunity is intended for therapyor enhanced antibody survival time. In general, HAMA responses pose apotential obstacle to realizing the full diagnostic and therapeuticpotential of murine anti-CD74 mAbs. Therefore, the development ofchimeric, humanized and human anti-CD74 mAbs and fragments thereof,antibody fusion proteins thereof and fragments thereof, immunoconjugatesfor therapy and diagnosis, multivalent and/or multispecific mAbs, andfragments thereof and vaccine conjugates thereof would be extremelyuseful for therapy and diagnosis, with reduced production of humananti-mouse antibodies.

SUMMARY OF THE INVENTION

The present invention is directed to anti-CD74 antibodies and fragmentsthereof and antibody fusion proteins thereof, particularly chimeric,humanized or human antibodies, which can be rapidly internalized into acell.

The present invention is further directed to anti-CD74 antibody fusionproteins containing antibodies or fragments thereof that are fused toeach other and/or to other antibodies and fragments thereof of thepresent invention.

The present invention additionally is directed to immunoconjugatescontaining the anti-CD74 antibodies or fragments thereof or the antibodyfusion proteins or fragments thereof of the present invention linked toa diagnostic or therapeutic agent.

The present invention also is directed to a vaccine comprising anantibody conjugate containing the anti-CD74 antibodies or fragmentsthereof or the antibody fusion proteins or fragments thereof of thepresent invention linked to antigenic peptides.

The present invention further is directed to a bispecific ormultispecific antibody comprising an antibody conjugate containing theanti-CD74 antibodies or fragments thereof or the antibody fusionproteins or fragments thereof of the present invention linked to anantibody or antibody fragment specific for a cancer marker substance, aneptitope on the surface of an infectious disease organism or a noxioussubstance in the blood or other body fluid.

The present invention is additionally directed to methods of treatingand diagnosing diseases using the CD74 antibodies and fragments thereofor antibody fusion proteins thereof and conjugates thereof of thepresent invention.

The present invention also is directed to DNA sequences encoding theCD74 antibodies or fragments thereof or antibody fusion proteins orfragments thereof, immunoconjugates and antibody conjugates andmultispecific antibodies thereof, expression vectors and host cellscontaining the DNA sequences, and methods of expressing these CD74antibodies of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the DNA and amino acid sequences of the murine LL1 heavyand light chain variable regions. FIG. 1A shows the DNA (SEQ ID NO:1)and amino acid (SEQ ID NO:2) sequences of the LL1 VH obtained by RT-PCR.FIG. 1B shows the DNA (SEQ ID NO:3) and amino acid (SEQ ID NO:4)sequences of the LL1Vk obtained by 5′-RACE. Amino acid sequences encodedby the corresponding DNA sequences are given as one-letter codes belowthe nucleotide sequence. Numbering of the nucleotide sequences is on theright side. The amino acid residues in the CDR regions are shown in boldand underlined. Kabat's Ig molecule numbering is used for amino acidresidues as shown by the numbering above the amino acid residues. Theresidues numbered by a letter following a particular digit indicates theinsertion residues defined by Kabat numbering scheme. The insertionresidues numbered with a letter have the same preceding digit. Forexample, residues 82A, 82B and 82C in FIG. 1A are indicated as 82A, B,and C.

FIG. 2 shows the DNA and amino acid sequences of the chimeric LL1 (cLL1)heavy and light chain variable regions expressed in Sp2/0 cells. FIG. 2Ashows the DNA (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequences ofthe cLL1VH. FIG. 2B shows the double-stranded DNA (SEQ ID NO:7) andamino acid (SEQ ID NO:8) sequences of the cLL1Vk. Amino acid sequencesencoded by the corresponding DNA sequences are given as one-lettercodes. The amino acid residues in the CDR regions are shown in bold andunderlined. The numbering of nucleotides and amino acids is same as thatin FIG. 1. The restriction sites used for constructing the cLL1 areboxed and indicated.

FIG. 3 shows the alignment of the amino acid sequences of light andheavy chain variable regions of a human antibody, cLL1 and hLL1. FIG. 3Ashows the VH amino acid sequence alignment of the human antibody RF-TS3,(SEQ ID NO:9) cLL1 (SEQ ID NO:10) and hLL1 (SEQ ID NO:11) and FIG. 3Bshows the Vk amino acid sequence alignment of the human antibodyHF-21/28, (SEQ ID NO:12) cLL1 (SEQ ID NO:13) and hLL1 (SEQ ID NO:14).Dots indicate the residues in cLL1 that are identical to thecorresponding residues in the human antibodies. Boxed regions prepresentthe CDR regions. Both N- and C-terminal residues (underlined) of cLL1are fixed by the staging vectors used and not compared with the humanantibodies. Kabat's Ig molecule number scheme is used as in FIG. 1.

FIG. 4 shows the DNA and amino acid sequences of the humanized LL1(hLL1) heavy and light chain variable regions expressed in Sp2/0 cells.FIG. 4A shows the DNA (SEQ ID NO:15) and amino acid (SEQ ID NO:16)sequences of the hLL1VH and FIG. 4B shows the DNA (SEQ ID NO:17) andamino acid (SEQ ID NO:18) sequences of the hLL1Vk. Amino acid sequencesencoded by the corresponding DNA sequences are given as one lettercodes. The amino acid residues in the CDR regions are shown in bold andunderlined. Kabat's Ig molecule numbering scheme is used for amino acidresidues as in FIG. 1A and FIG. 1B.

FIG. 5 shows a schematic diagram of construction of hLL1VH gene. Oligosused as templates and primers are shown as arrow lines. The arrow headsindicate the 3′-ends. The sense DNA strands (templates, primers and PCRproducts) are shown in solid lines and the anti-sense strands in dotedlines. The Vk gene was similarly constructed.

FIG. 6 shows the result of a competitive cell surface binding assay tocompare the binding affinity of cLL1 with that of murine LL1. Varyingconcentrations of cLL1 (triangles) or mLL1 (diamonds) were mixed with aconstant amount of ¹²⁵I-labeled mLL1 and incubated with Raji cells at 4°C. for 1 h. The cell surface bound radiolabeled mLL1 was counted afterwashing. cLL1 and the murine LL1 competed equally well for the bindingof radiolabeled LL1 to Raji cells, confirming the cloned V genes areauthentic.

FIG. 7 shows the result of a competitive binding assay in Raji cellmembrane coated micro wells to compare the binding affinity of hLL1 withthat of cLL1. Varying concentrations of hLL1 (triangles) or cLL1(diamonds) were mixed with a constant amount of HRP conjugated LL1 andincubated in 96-well microtitration plate coated with Raji membraneextracts at room temperature for 1 h. The membrane bound HRP-LL1 wasmeasured. hLL1 and cLL1 competed equally well for the binding ofHRP-LL1, indicating the binding specificity and affinity of mAb LL1 arepreserved in the humanized LL1.

FIG. 8 shows the fate of ¹²⁵I-labeled hLL1 and mLL1 bound to the surfaceof Raji cells. The radiolabeled hLL1 (solid line with symbols) or mLL1(dotted line with symbols) was incubated with Raji cells and unbound Abswere removed by washing. The cells were then cultured as normal and theradiolabeled Abs associated with cells (diamond lines), secreted intomedium (triangle lines) or degraded (circle lines) were measured atindicated time point. FIG. 8A shows the fate of the bound Ab followedfor up to 3 days. FIG. 8B shows the result of hLL1 processing studied atearly time points (less than 3 h). The data was averaged of twoexperiments.

FIG. 9 shows the cytotoxicity effect of crosslinked LL1 Abs on Rajicells. 5×10⁵ Raji cells were seeded at day 0 in 1 ml of culture mediumcontaining (as indicated on top of the panels) 5 μg/ml of mLL1, cLL1 orhLL1, or no any Ab (Nil), with 50 μg/ml of -mFc or -hFc Ab, or withoutany crosslinker (Nil), indicated at right side of panels. The numbers oftotal and viable cells were counted daily for 3 days. Percentage ofviable cells (squares) and the ratio of viable cells over the viablecells at time zero (diamonds) were calculated and plotted againstculture time.

FIG. 10 shows the cytotoxicity effect of crosslinked hLL1 on Daudicells. 5×10⁵ Daudi cells were seeded at day 0 in 1 ml of culture mediumcontaining (as indicated on top of the panels) 5 μg/ml of hLL1, or hLL2(an anti-CD22, internalizing Ab), or no any Ab (Nil), with 50 μg/ml of-hFc Ab, or without (Nil), indicated at right side of panels. Thenumbers of total and viable cells were counted daily for 3 days.Percentage of viable cells (squares) and the ratio of viable cells overthe viable cells at time zero (diamonds) were calculated and plottedagainst culture time.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, the terms “a” or “an” mean “one or more.”

Overview

The present invention provides a humanized, a chimeric and a humananti-CD74 mAb, fragments thereof, an antibody fusion protein, andtherapeutic and diagnostic conjugates thereof useful for treatment ofmammalian subjects, humans and domestic animals, alone, as a conjugateor administered in combination with other therapeutic agents, includingother naked antibodies and antibody therapeutic conjugates as part of amultimodal therapy regimen. Methods of treatment and diagnosis of B-cellmalignancies, other CD74 positive malignancies and autoimmune diseasesare disclosed.

Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-CD74 monoclonal antibody fragmentbinds with an epitope of CD74. The term “antibody fragment” alsoincludes any synthetic or genetically engineered protein that acts likean antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), and minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector functions, such as complementfixation and ADCC (antibody dependent cell cytotoxicity) that setmechanisms into action that may result in cell lysis. However, it ispossible that the Fc portion is not required for therapeutic function,with other mechanisms, such as apoptosis, coming into play. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric, humanized or humanantibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as acat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

100351 Human antibodies may also be generated by in vitro activated Bcells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incoporatedin their entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a subfragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, enzymes, nucleases, hormones, immunomodulators, antisenseoligonucleotides, chelators, boron compounds, photoactive agents or dyesand radioisotopes.

A diagnostic agent is a molecule or atom which is administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing a disease by locating thecells containing the antigen. Useful diagnostic agents include, but arenot limited to, radioisotopes, dyes (such as with thebiotin-streptavidin complex), contrast agents, fluorescent compounds ormolecules and enhancing agents (e.g., paramagnetic ions) for magneticresonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI techniqueand the preparation of antibodies conjugated to a MRI enhancing agentand is incorporated in its entirety by reference. Preferably, thediagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents for use in magnetic resonance imaging,and fluorescent compounds. In order to load an antibody component withradioactive metals or paramagnetic ions, it may be necessary to react itwith a reagent having a long tail to which are attached a multiplicityof chelating groups for binding the ions. Such a tail can be a polymersuch as a polylysine, polysaccharide, or other derivatized orderivatizable chain having pendant groups to which can be boundchelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crownethers, bis-thiosemicarbazones, polyoximes, and like groups known to beuseful for this purpose. Chelates are coupled to the peptide antigensusing standard chemistries. The chelate is normally linked to theantibody by a group which enables formation of a bond to the moleculewith minimal loss of immunoreactivity and minimal aggregation and/orinternal cross-linking. Other, more unusual, methods and reagents forconjugating chelates to antibodies are disclosed in U.S. Pat. No.4,824,659 to Hawthorne, entitled “Antibody Conjugates”, issued Apr. 25,1989, the disclosure of which is incorporated herein in its entirety byreference. Particularly useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used withdiagnostic isotopes in the general energy range of 60 to 4,000 keV, suchas ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²CU, ⁶⁴CU, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc,^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radio-imaging. The same chelates,when complexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI, when used along with the antibodies ofthe invention. Macrocyclic chelates such as NOTA, DATA, and TETA are ofuse with a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for RAIT are encompassed by the invention.

An immunoconjugate is a conjugate of an antibody component with atherapeutic or diagnostic agent. The diagnostic agent can comprise aradioactive or non-radioactive label, a contrast agent (such as formagnetic resonance imaging, computed tomography or ultrasound), and theradioactive label can be a gamma-, beta-, alpha-, Auger electron-, orpositron-emitting isotope.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, such as bacteria, yeastand mammalian cells, as well as an transgenic animal, that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell or cells of the host cells. Suitablemammalian host cells include myeloma cells, such as SP2/O cells, and NSOcells, as well as Chinese Hamster Ovary (CHO) cells, hybridoma celllines and other mammalian host cell useful for expressing antibodies.Also particularly useful to express mAbs and other fusion proteins, is ahuman cell line, PER.C6 disclosed in WO 0063403 A2, which produces 2 to200-fold more recombinant protein as compared to conventional mammaliancell lines, such as CHO, COS, Vero, Hela, BHK and SP2-cell lines.Special transgenic animals with a modified immune system areparticularly useful for making fully human antibodies.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent single-chain antibody or antibody fragment segments with thesame or different specificities are linked. Valency of the fusionprotein indicates how many binding arms or sites the fusion protein hasto a single antigen or epitope; i.e., monovalent, bivalent, trivalent ormutlivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindswith one epitope, for example a diabody with two binding site reactivewith the same antigen. The fusion protein may comprise a single antibodycomponent, a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity would be for aB-cell, T-cell, myeloid-, plasma-, and mast-cell antigen or epitope.Another specificity could be to a different antigen on the same celltype, such as CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, and CD22on B-cells. Multispecific, multivalent antibodies are constructs thathave more than one binding site, and the binding sites are of differentspecificity. For example, a diabody, where one binding site reacts withone antigen and the otherwith another antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, a B-cell, T-cell, myeloid-, plasma-,and mast-cell antigen or epitope and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering. In one form, the bispecific fusionprotein is monovalent, consisting of, for example, a scFv with a singlebinding site for one antigen and a Fab fragment with a single bindingsite for a second antigen. In another form, the bispecific fusionprotein is divalent, consisting of, for example, an IgG with a bindingsite for one antigen and two scFv with two binding sites for a secondantigen.

Caninized or felinized antibodies are recombinant proteins in whichrodent (or another species) complementarity determining regions of amonoclonal antibody have been transferred from heavy and light variablechains of rodent (or another species) immunoglobulin into a dog or cat,respectively, immunoglobulin variable domain.

Domestic animals include large animals such as horses, cattle, sheep,goats, llamas, alpacas, and pigs, as well as companion animals. In apreferred embodiment, the domestic animal is a horse.

Companion animals include animals kept as pets. These are primarily dogsand cats, although small rodents, such as guinea pigs, hamsters, rats,and ferrets, are also included, as are subhuman primates such asmonkeys. In a preferred embodiment the companion animal is a dog or acat.

Preparation of Monoclonal Antibodies Including Chimeric, Humanized andHuman Antibodies

Monoclonal antibodies (MAbs) are a homogeneous population of antibodiesto a particular antigen and the antibody comprises only one type ofantigen binding site and binds to only one epitope on an antigenicdeterminant. Rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art. See, for example,Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.),CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley &Sons 1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies canbe obtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (1gG), “in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Techniques for constructing chimeric antibodies arewell known to those of skill in the art. As an example, Leung et al.,Hybridoma 13:469 (1994), describe how they produced an LL2 chimera bycombining DNA sequences encoding the VK and VH domains of LL2 monoclonalantibody, an anti-CD22 antibody, with respective human kappa and IgG₁constant region domains. This publication also provides the nucleotidesequences of the LL2 light and heavy chain variable regions, VK and VH,respectively. Techniques for producing humanized MAbs are described, forexample, by Jones et al., Nature 321: 522 (1986), Riechmann et al.,Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988),Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit.Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844(1993), each of which is hereby incorporated by reference.

To this end, the present invention describes chimeric, humanized andhuman antibodies and fragments thereof that bind the CD74 antigen andcan be used for diagnostic and therapeutic methods. Humanized antibodiesand antibody fragments are described in Provisional U.S. Applicationtitled “Anti-CD20 Antibodies And Fusion Proteins Thereof And Methods OfUse”, Attorney Docket No. 18733/1073, U.S. Provisional No. 60/356,132,U.S. Provisional Application No. 60/416,232 and Attorney Docket No.18733/1155; hMN-14 antibodies, such as those disclosed in U.S. Pat. No.5,874,540, which is a Class III anti-carcinoembryonic antigen antibody(anti-CEA antibody); Mu-9 antibodies, such as those described in U.S.application Ser. No. 10/116,116; AFP antibodies, such as those describedin U.S. Provisional Application No. 60/399,707; PAM4 antibodies, such asthose described in Provisional U.S. Application titled “MonoclonalAntibody cPAM4”, Attorney Docket No. 18733/1102; RS7 antibodies, such asthose described in U.S. Provisional Application No. 60/360,229; and CD22antibodies, such as those disclosed in U.S. Pat. Nos. 5,789,554 and6,187,287 and U.S. application Ser. Nos. 09/741,843 and 09/988,013, allof which are incorporated herein by reference in their entirety.

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimeric mAbwith one or more different human FR. Specifically, mouse CDRs aretransferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239:1534 (1988). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, W0 0029584A1.

Another method for producing the antibodies of the present invention isby production in the milk of transgenic livestock. See, e.g., Colman,A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690,both of which are incorporated in their entirety by reference. Two DNAconstructs are prepared which contain, respectively, DNA segmentsencoding paired immunoglobulin heavy and light chains. The DNA segmentsare cloned into expression vectors which contain a promoter sequencethat is preferentially expressed in mammary epithelial cells. Examplesinclude, but are not limited to, promoters from rabbit, cow and sheepcasein genes, the cow a-lactoglobulin gene, the sheep β-lactoglobulingene and the mouse whey acid protein gene. Preferably, the insertedfragment is flanked on its 3′ side by cognate genomic sequences from amammary-specific gene. This provides a polyadenylation site andtranscript-stabilizing sequences. The expression cassettes arecoinjected into the pronuclei of fertilized, mammalian eggs, which arethen implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of bothtransgenes by Southern analysis. In order for the antibody to bepresent, both heavy and light chain genes must be expressed concurrentlyin the same cell. Milk from transgenic females is analyzed for thepresence and functionality of the antibody or antibody fragment usingstandard immunological methods known in the art. The antibody can bepurified from the milk using standard methods known in the art.

A fully human antibody of the present invention, i.e., human anti-CD74MAbs or other human antibodies, such as anti-CD22, anti-CD19, anti-CD23,anti-CD20 or anti-CD21 MAbs for combination therapy with humanized,chimeric or human anti-CD74 antibodies, can be obtained from atransgenic non-human animal. See, e.g., Mendez et al., Nature Genetics,15: 146-156 (1997); U.S. Pat. No. 5,633,425, which are incorporated intheir entirety by reference. For example, a human antibody can berecovered from a transgenic mouse possessing human immunoglobulin loci.The mouse humoral immune system is humanized by inactivating theendogenous immunoglobulin genes and introducing human immunoglobulinloci. The human immunoglobulin loci are exceedingly complex and comprisea large number of discrete segments which together occupy almost 0.2% ofthe human genome. To ensure that transgenic mice are capable ofproducing adequate repertoires of antibodies, large portions of humanheavy- and light-chain loci must be introduced into the mouse genome.This is accomplished in a stepwise process beginning with the formationof yeast artificial chromosomes (YACs) containing either human heavy- orlight-chain immunoglobulin loci in germline configuration. Since eachinsert is approximately 1 Mb in size, YAC construction requireshomologous recombination of overlapping fragments of the immunoglobulinloci. The two YACs, one containing the heavy-chain loci and onecontaining the light-chain loci, are introduced separately into mice viafusion of YAC-containing yeast spheroblasts with mouse embryonic stemcells. Embryonic stem cell clones are then microinjected into mouseblastocysts. Resulting chimeric males are screened for their ability totransmit the YAC through their germline and are bred with mice deficientin murine antibody production. Breeding the two transgenic strains, onecontaining the human heavy-chain loci and the other containing the humanlight-chain loci, creates progeny which produce human antibodies inresponse to immunization.

Further recent methods for producing bispecific mAbs include engineeredrecombinant mAbs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10 (10): 1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bispecific fusion proteins canbe produced using molecular engineering. In one form, the bispecificfusion protein is monovalent, consisting of, for example, a scFv with asingle binding site for one antigen and a Fab fragment with a singlebinding site for a second antigen. In another form, the bispecificfusion protein is divalent, consisting of, for example, an IgG with twobinding sites for one antigen and two scFv with two binding sites for asecond antigen.

Bispecific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.Recombinant methods can be used to produce a variety of fusion proteins.For example, a fusion protein comprising a Fab fragment derived from ahumanized monoclonal anti-CD74 antibody and a scFv derived from a murineanti-diDTPA can be produced. A flexible linker, such as GGGS connectsthe scFv to the constant region of the heavy chain of the anti-CD74antibody. Alternatively, the scFv can be connected to the constantregion of the light chain of another humanized antibody. Appropriatelinker sequences necessary for the in-frame connection of the heavychain Fd to the scFv are introduced into the Vλ and Vκ domains throughPCR reactions. The DNA fragment encoding the scFv is then ligated into astaging vector containing a DNA sequence encoding the CH1 domain. Theresulting scFvCH1 construct is excised and ligated into a vectorcontaining a DNA sequence encoding the VH region of an anti-CD74antibody. The resulting vector can be used to transfect an appropriatehost cell, such as a mammalian cell for the expression of the bispecificfusion protein.

Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, Fab, Fv, sFv and the like. Otherantibody fragments include, but are not limited to: the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fab′expression expression libraries can be constructed (Huse et al., 1989,Science, 246: 1274-1281) to allow rapid and easy identification ofmonoclonal Fab′ fragments with the desired specificity. The presentinvention encompasses antibodies and antibody fragments.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,“Single Chain Antibody Variable Regions,” TIBTECH, Vol 9: 132-137(1991). These references are incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Anti-CD74 Antibodies

The anti-CD74 mAbs of the present invention contain specific murine CDRsthat have specificity for the CD74 antigen. The anti-CD74 mAbs of thepresent invention are humanized, chimeric or human mAbs and they containthe amino acids of the CDRs of a murine anti-CD74 mAb, the murine LL1mAb. The humanized anti-CD74 monoclonal antibody (mAb) or fragmentthereof comprise CDRs of a light chain variable region of a murineanti-CD74 mAb, that comprises CDR1 comprising an amino acid sequenceRSSQSLVHRNGNTYLH (SEQ ID NO:19), CDR2 comprising an amino acid sequenceTVSNRFS (SEQ ID NO:20), and CDR3 comprising an amino acid sequenceSQSSHVPPT (SEQ ID NO:21). Further, the humanized anti-CD74 monoclonalantibody or fragment thereof comprises the heavy chain variable regionof said humanized mAb that comprises CDRs of a heavy chain variableregion of a murine anti-CD74 mAb, that comprises CDR1 comprising anamino acid sequence NYGVN (SEQ ID NO:22), CDR2 comprising an amino acidsequence WINPNTGEPTFDDDFKG (SEQ ID NO:23), and CDR3 comprising an aminoacid sequence SRGKNEAWFAY (SEQ ID NO:24). Further, the humanized mAbretains substantially the specificity for the CD74, i.e., the MHCclass-II invariant chain, Ii, present on the surface of cells, such asB-lymphocytes, monocyte and histiocytes, as well as B-cell lymphoma andleukemia, as well as myeloma cells resulting in the rapidinternalization and catabolization of these mAbs, fragments thereof ormAb conjugates.

In one embodiment, a CD74 antibodies of the present invention is ahumanized anti-CD74 monoclonal antibody (mAb) or fragment thereofcomprising light and heavy chain variable regions comprisingcomplementarity-determining regions (CDRs) of murine anti-CD74 (mLL1)and the framework (FR) regions of a human antibody, wherein the lightchain variable region of the humanized mAb comprises CDRs of a lightchain variable region of a murine anti-CD74 mAb, that comprises CDR1comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO:19), CDR2comprising an amino acid sequence TVSNRFS (SE ID NO: 20), and CDR3comprising an amino acid sequence SQSSHVPPT (SEQ ID NO:21), and whereinthe heavy chain variable region of the humanized mAb comprises CDRs of aheavy chain variable region of a murine anti-CD74 mAb, that comprisesCDR1 comprising an amino acid sequence NYGVN (SEQ ID NO:22), CDR2comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO:23), andCDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO:24). Themurine CDRs of the heavy and light chain variable regions are shown inFIGS. 1A and 1B, respectively. The human FRs of the light and heavychain variable regions may be modified to maintain specificity to CD74by substituting at least one amino acid substituted from thecorresponding FRs of the murine mAb. More specifically, one or morespecific amino acids from the murine mAb identified by amino acidresidue 2, 3, 4, 46, 87 and 100 of the murine light chain variableregion of the cLL1Vk sequence of FIG. 3B, and amino acid residues 5, 37,38, 46, 68, 91 and 93 of the murine heavy chain variable region of thecLL1VH sequence of FIG. 3A may be maintained in the human FRs of thehumanized anti-CD74 to maintain specificity.

In a preferred embodiment, the humanized anti-CD74 mAb, the humanizedLL1 (hLL1) or fragment thereof containing a heavy chain variable regionof FIG. 4A and a light chain variable region of FIG. 4B is used in themethods disclosed in the present invention. More specifically, thehumanized anti-CD74 mAb or fragment thereof contains a light and heavychain constant region of a human antibody or a portion thereofAdditionally, the humanized anti-CD74 mAb or fragment thereof of anyoneof the humanized anti-CD74 mAbs or fragments thereof, described herein,can be a humanized IgG1.

Although humanized anti-CD74 mAbs are preferred, chimeric anti-CD74(cCD74) mAbs or fragments thereof also are encompassed by the presentinvention. In one embodiment, the chimeric anti-CD74 monoclonalantibody, (mAb) or fragment thereof comprises a light chain variableregion of a murine anti-CD74 mAb, that comprises CDR1 comprising anamino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO:19), CDR2 comprising anamino acid sequence TVSNRFS (SEQ ID NO:20), and CDR3 comprising an aminoacid sequence SQSSHVPPT (SEQ ID NO:21). In a further embodiment, thechimeric anti-CD74 monoclonal antibody or fragment thereof comprises aheavy chain variable region of a murine anti-CD74 mAb, that comprisesCDR1 comprising an amino acid sequence NYGVN (SEQ ID NO:22), CDR2comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO:23), andCDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO:24). In afurther embodiment the chimeric anti-CD74 mAb comprises light and heavychain variable regions comprising complementarity-determining regions(CDRs) of a murine anti-CD74 mAb and the framework (FR) regions of amurine anti-CD74 mAb and the light and heavy chain constant regions of ahuman antibody, wherein the light chain variable region of the chimericmAb comprises CDRs of a light chain variable region of a murine antiCD74mAb, that comprises CDR1 comprising an amino acid sequenceRSSQSLVHRNGNTYLH (SEQ ID NO:19), CDR2 comprising an amino acid sequenceTVSNRFS (SEQ ID NO:20), and CDR3 comprising an amino acid sequenceSQSSHVPPT (SEQ ID NO:21), and wherein the heavy chain variable region ofsaid chimeric mAb comprises CDRs of a heavy chain variable region of amurine anti-CD74 mAb, that comprises CDR1 comprising an amino acidsequence NYGVN (SEQ ID NO:22), CDR2 comprising an amino acid sequenceWINPNTGEPTFDDDFKG (SEQ ID NO:23), and CDR3 comprising an amino acidsequence SRGKNEAWFAY (SEQ ID NO:24). The preferred chimeric anti-CD74mAb or fragment thereof comprises a heavy chain variable region of FIG.2A and a light chain variable region of FIG. 2B.

Also encompassed within the present invention is a human anti-CD74monoclonal antibody (mAb) or fragment thereof comprising a light chainvariable region of the human anti-CD74 mAb that comprises CDR1comprising an amino acid sequence RSSOSLVHRNGNTYLH (SEQ ID NO:19), CDR2comprising an amino acid sequence TVSNRFS (SEQ ID NO:20), and CDR3comprising an amino acid sequence SQSSHVPPT (SEQ ID NO:21). Further,encompasses is a human anti-CD74 monoclonal antibody (mAb) or fragmentthereof comprising a heavy chain variable region of said human mAb thatcomprises CDRs of a heavy chain variable region of a murine anti-CD74mAb, that comprises CDR1 comprising an amino acid sequence NYGVN (SEQ IDNO:22), CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ IDNO:23), and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ IDNO:24). More preferably, the present invention discloses a humananti-CD74 (huCD74) monoclonal antibody (mAb) or fragment thereofcomprising the light and heavy chain variable and constant regions of ahuman antibody, wherein the huCD74 CDRs of the light chain variableregion of the human anti-CD74 mAb comprises CDR1 comprising an aminoacid sequence RSSOSLVHRNGNTYLH, CDR2 comprising an amino acid sequenceTVSNRFS, and CDR3 comprising an amino acid sequence SQSSHVPPT, andwherein the heavy chain variable region of the human mAb comprises CDRsof a heavy chain variable region of a murine anti-CD74 mAb, thatcomprises CDR1 comprising an amino acid sequence NYGVN, CDR2 comprisingan amino acid sequence WINPNTGEPTFDDDFKG, and CDR3 comprising an aminoacid sequence SRGKNEAWFAY.

Each of the human, chimeric or humanized anti-CD74 mAb of the presentinvention is preferably an IgG1, where the constant regions arepreferably a human IgG1, but the IgG1 may be referred to as a humanIgG1, a chimeric IgG1 or a humanized IgG1, respectively. In particular,the humanized CD74 mAb, hLL1, has constant domains and the hinge regionfrom a human IgG1. Preferably, both the chimeric and the human LL1 mAbhas the same constant domain and hinge region. However, modificationscan be made so that the constant regions of the IgG1 are replaced withwith human constant regions of human IgG2a, IgG3 or IgG4.

The present invention also is directed to a murine anti-CD74 monoclonalantibody or fragment thereof, comprising CDRs of a light chain variableregion of a murine anti-CD74 mAb, that comprises CDR1 comprising anamino acid sequence RSSQSLVHRNGNTYLH (SEQ ID NO:19), CDR2 comprising anamino acid sequence TVSNRFS (SEQ ID NO:20), and CDR3 comprising an aminoacid sequence SQSSHVPPT (SEQ ID NO:21). Further, the murine antiCD74monoclonal antibody or fragment thereof, comprising CDRs of a heavychain variable region of a murine anti-CD74 mAb, that comprises CDR1comprising an amino acid sequence NYGVN (SEQ ID NO:22), CDR2 comprisingan amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO:23), and CDR3comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO:24). Morepreferably, the murine anti-CD74 monoclonal antibody or fragment thereofcomprising complementarity-determining regions (CDRs) of murineanti-CD74 (mLL1) and the framework (FR) regions of a murine anti-CD74antibody, wherein the light chain variable region of said murine mAbcomprises CDR1 comprising an amino acid sequence RSSQSLVHRNGNTYLH (SEQID NO:19), CDR2 comprising an amino acid sequence TVSNRFS (SEQ IDNO:20), and CDR3 comprising an amino acid sequence SQSSHVPPT (SEQ IDNO:21), and wherein the heavy chain variable region of said murine mAbcomprises CDR1 comprising an amino acid sequence NYGVN (SEQ ID NO:22),CDR2 comprising an amino acid sequence WINPNTGEPTFDDDFKG (SEQ ID NO:22),and CDR3 comprising an amino acid sequence SRGKNEAWFAY (SEQ ID NO:24).

Each of the human, chimeric, humanized or murine anti-CD74 mAbs orfragment thereof of the present invention possess at least one of thefollowing properties: it binds specifically and is reactive with theantigen, CD74, its binding to CD74 is blocked by an antibody or fragmentthereof specific for or reactive with CD74; it is internalized by Rajilymphoma cells in culture; and it induces apoptosis of Raji cells incell culture when cross-linked with goat antisera reactive with the Fcof a murine IgG1 mAb.

The fragments of the human, chimeric or humanized anti-CD74 mAb may be afragment, such is F(ab′)₂, Fab, scFv, Fv, or a fusion constructutilizing part or all the light and heavy chains of the F(ab′)2, Fab,scFv,or Fv. It is important that the fragment binds to CD74.

Multispecific and Multivalent Antibodies

The anti-CD74 antibodies, as well as other antibodies with differentspecificities for use in combination therapy, described herein, can alsobe made as multispecific antibodies (comprising at least one bindingsite to a CD74 epitope or antigen and at least one binding site toanother epitope on CD74 or another antigen) and multivalent antibodies(comprising mutliple binding sites to the same epitope or antigen), orthe antibodies can be both multivalent and multispecific.

A preferred antibody fusion protein of the present invention containsfour or more Fvs, or Fab's of the humanized, chimeric, human or murineanti-CD74 mAbs or fragments thereof described herein. Additionally,another preferred antibody fusion protein contains one or more Fvs, orFab's of the mAbs or fragments thereof of the humanized, chimeric, humanor murine anti-CD74 mAbs or fragments thereof described herein, and oneor more Fvs or Fab's from antibodies specific for another antigen thatis specific for a tumor cell marker that is not a CD74 antigen, that isexpressed by the CD74-expressing cells, such as, for example, a tumormarker selected from a B-cell lineage antigen, such as CD 19, CD20, orCD22 for the treatment of B-cell malignancies; as well as other CD74positive cells causing other types of malignancies, such as S100 inmelanoma, etc. Further, the tumor cell marker may be a non-B-celllineage antigen selected from the group consisting of HLA-DR, CD30,CD33, CD52 MUC1 and TAC.

The present invention also provides a bispecific or multispecificantibody, wherein the anti-CD74 mAbs or fragments thereof or antibodyfusion proteins thereof of the present invention are linked to anantibody or antibody fragment specific for a cancer marker substance, anepitope on the surface of a infectious disease organism, or a noxioussubstance in the blood or other body fluids. The bispecific andmultispecific antibodies of the present invention are particularlyuseful in the method of inducing clearance of a variety of noxioussubstances, where the bispecific antibody has at least one specificityfor a noxious substance, such as a pathogenic organism, and at least onespecificity for CD74, the HLA class-II invariant chain (Ii), asdescribed in detail in U.S. Ser. No. 09/314,135, filed on May 19, 1999,entitled “Therapeutic Using a Bispecific Antibody,” which is hereinincorporated in its entirety by reference.

The present invention further provides a bispecific antibody or antibodyfragment having at least a binding region that specifically binds atargeted cell marker and at least one other binding region thatspecifically binds a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment.

A variety of recombinant methods can be used to produce bispecificantibodies and antibody fragments as described above.

An anti-CD74 multivalent antibody is also contemplated in the presentinvention. This multivalent target binding protein is constructed byassociation of a first and a second polypeptide. The first polypeptidecomprises a first single chain Fv molecule covalently linked to a firstimmunoglobulin-like domain that preferably is an immunoglobulin lightchain variable region domain. The second polypeptide comprises a secondsingle chain Fv molecule covalently linked to a secondimmunoglobulin-like domain tht preferably is an immunoglobulin heavychain variable region domain. Each of the first and second single chainFv molecules forms a target binding site, and the first and secondimmunoglobulin-like domains associate to form a third target bindingsite.

A single chain Fv molecule with the VL-L-VH configuration, wherein L isa linker, may associate with another single chain Fv molecule with theVH-L-VL configuration to form a bivalent dimer. In this case, the VLdomain of the first scFv and the VH domain of the second scFv moleculeassociate to form one target binding site, while the VH domain of thefirst scFv and the VL domain of the second scFv associate to form theother target binding site.

Another embodiment of the present invention is a CD74 bispecific,trivalent targeting protein comprising two heterologous polypeptidechains associated non-covalently to form three binding sites, two ofwhich have affinity for one target and a third which has affinity for ahapten that can be made and attached to a carrier for a diagnosticand/or therapeutic agent. Preferably, the binding protein has two CD20binding sites and one CD22 binding site. The bispecific, trivalenttargeting agents have two different scFvs, one scFv contains two V_(H)domains from one antibody connected by a short linker to the VL domainof another antibody and the second scFv contains two VL domains from thefirst antibody connected by a short linker to the VH domain of the otherantibody. The methods for generating multivalent, multispecific agentsfrom VH and VL domains provide that individual chains synthesized from aDNA plasmid in a host organism are composed entirely of VH domains (theVH-chain) or entirely of VL domains (the VL-chain) in such a way thatany agent of multivalency and multispecificity can be produced bynon-covalent association of one VH-chain with one VL-chain. For example,forming a trivalent, trispecific agent, the VH-chain will consist of theamino acid sequences of three V_(H) domains, each from an antibody ofdifferent specificity, joined by peptide linkers of variable lengths,and the VL-chain will consist of complementary VL domains, joined bypeptide linkers similar to those used for the VH-chain. Since the VH andVL domains of antibodies associate in an anti-parallel fashion, thepreferred method in this invention has the VL domains in the VL-chainarranged in the reverse order of the VH domains in the VH-chain.

Diabodies, Triabodies and Tetrabodies

The anti-CD74 antibodies of the present invention can also be used toprepare functional bispecific single-chain antibodies (bscAb), alsocalled diabodies, and can be produced in mammalian cells usingrecombinant methods. See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92:7021-7025, 1995, incorporated. For example, bscAb are produced byjoining two single-chain Fv fragments via a glycine-serine linker usingrecombinant methods. The V light-chain (VL) and V heavy-chain (VH)domains of two antibodies of interest are isolated using standard PCRmethods. The VL and VH cDNA's obtained from each hybridoma are thenjoined to form a single-chain fragment in a two-step fusion PCR. Thefirst PCR step introduces the (Gly₄-Ser₁)₃ linker (SEQ ID NO:25), andthe second step joins the VL and VH amplicons. Each single chainmolecule is then cloned into a bacterial expression vector. Followingamplification, one of the single-chain molecules is excised andsub-cloned into the other vector, containing the second single-chainmolecule of interest. The resulting bscAb fragment is subcloned into aneukaryotic expression vector. Functional protein expression can beobtained by transfecting the vector into Chinese Hamster Ovary cells.Bispecific fusion proteins are prepared in a similar manner. Bispecificsingle-chain antibodies and bispecific fusion proteins are includedwithin the scope of the present invention.

For example, a humanized, chimeric or human anti-CD74 monoclonalantibody can be used to produce antigen specific diabodies, triabodies,and tetrabodies. The monospecific diabodies, triabodies, and tetrabodiesbind selectively to targeted antigens and as the number of binding siteson the molecule increases, the affinity for the target cell increasesand a longer residence time is observed at the desired location. Fordiabodies, the two chains comprising the VH polypeptide of the humanizedCD74 mAb connected to the VK polypeptide of the humanized CD74 mAb by afive amino acid residue linker are utilized. Each chain forms one halfof the humanized CD74 diabody. In the case of triabodies, the threechains comprising V_(H) polypeptide of the humanized CD74 MAb connectedto the VK polypeptide of the humanized CD74 MAb by no linker areutilized. Each chain forms one third of the hCD74 triabody.

The ultimate use of the bispecific diabodies described herein is forpretargeting CD74 positive tumors for subsequent specific delivery ofdiagnostic or therapeutic agents. These diabodies bind selectively totargeted antigens allowing for increased affinity and a longer residencetime at the desired location. Moreover, non-antigen bound diabodies arecleared from the body quickly and exposure of normal tissues isminimized. The diagnostic and therapeutic agents can include isotopes,drugs, toxins, cytokines, hormones, enzymes, oligonucleotides, growthfactors, conjugates, radionuclides, and metals. For example, gadoliniummetal is used for magnetic resonance imaging (MRI). Examples ofradionuclides are ²²⁵Ac, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In, ¹³¹I, ¹²⁵I,¹²³I, ^(99m)Tc, ^(94m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi,²¹³Bi, ³²P, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, and ²¹¹At. Other radionuclides are alsoavailable as diagnostic and therapeutic agents, especially those in theenergy range of 60 to 4,000 keV for diagnostic agents and in the energyrange of 60-700 for the therapeutic agents.

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius et al., Cancer Research(2000) 60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

Conjugated Multivalent and Multispecific Anti-CD74 Antibodies

In another embodiment of the instant invention is a conjugatedmultivalent anti-CD74 antibody. Additional amino acid residues may beadded to either the N- or C-terminus of the first or the secondpolypeptide. The additional amino acid residues may comprise a peptidetag, a signal peptide, a cytokine, an enzyme (for example, a pro-drugactivating enzyme), a hormone, a peptide toxin, such as pseudomonasextoxin, a peptide drug, a cytotoxic protein or other functionalproteins. As used herein, a functional protein is a protein that has abiological function.

In one embodiment, drugs, toxins, radioactive compounds, enzymes,hormones, cytotoxic proteins, chelates, cytokines and other functionalagents may be conjugated to the multivalent target binding protein,preferably through covalent attachments to the side chains of the aminoacid residues of the multivalent target binding protein, for exampleamine, carboxyl, phenyl, thiol or hydroxyl groups. Various conventionallinkers may be used for this purpose, for example, diisocyanates,diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.Conjugation of agents to the multivalent protein preferably does notsignificantly affect the protein's binding specificity or affinity toits target. As used herein, a functional agent is an agent which has abiological function. A preferred functional agent is a cytotoxic agent.

In still other embodiments, bispecific antibody-directed delivery oftherapeutics or prodrug polymers to in vivo targets can be combined withbispecific antibody delivery of radionuclides, such that combinationchemotherapy and radioimmunotherapy is achieved. Each therapy can beconjugated to the targetable conjugate and administered simultaneously,or the nuclide can be given as part of a first targetable conjugate andthe drug given in a later step as part of a second targetable conjugate.

In another embodiment, cytotoxic agents may be conjugated to a polymericcarrier, and the polymeric carrier may subsequently be conjugated to themultivalent target binding protein. For this method, see Ryser et al.,Proc. Natl. Acad. Sci. USA, 75:3867-3870, 1978, U.S. Pat. No. 4,699,784and U.S. Pat. No. 4,046,722, which are incorporated herein by reference.Conjugation preferably does not significantly affect the bindingspecificity or affinity of the multivalent binding protein.

Humanized, Chimeric and Human Antibodies use for Treatment and Diagnosis

Humanized, chimeric and human monoclonal antibodies, i.e., anti-CD74mAbs and other MAbs described herein, in accordance with this inventionare suitable for use in therapeutic methods and diagnostic methods.Accordingly, the present invention contemplates the administration ofthe humanized, chimeric and human antibodies of the present inventionalone as a naked antibody or administered as a multimodal therapy,temporally according to a dosing regimen, but not conjugated to, atherapeutic agent. An immunoconjugate is a conjugate comprising anantibody component comprising at least one mAb or fragment thereof orantibody fusion protein thereof of the humanized, chimeric or human CD74mAbs described in the present invention that binds to CD74, which islinked to a diagnostic or therapeutic agent.

The efficacy of the naked anti-CD74 mAbs can be enhanced bysupplementing naked antibodies with one or more other naked antibodies,i.e., mAbs to specific antigens, such as CD4, CD5, CD8, CD14, CD15,CD19, CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD46,CD52, CD54, CD8O, CD126, B7, MUC1, la, tenascin, HM1.24, or HLA-DR,preferably mature HLA-DR dimer, with one or more immunoconjugates ofanti-CD74, or antibodies to these recited antigens, conjugated withtherapeutic agents, including drugs, toxins, immunomodulators, hormones,enzymes, therapeutic radionuclides, etc., with one or more therapeuticagents, including drugs, toxins, immunomodulators, hormones, enzymes,therapeutic radionuclides, etc., administered concurrently orsequentially or according to a prescribed dosing regimen, with the mAbs.Preferred B-cell associated antigens include those equivalent to humanCD19, CD20, CD21, CD22, CD23, CD46, CD52, CD74, CD8O, and CD5 antigens.Preferred T-cell antigens include those equivalent to human CD4, CD8 andCD25 (the IL-2 receptor) antigens. An equivalent to HLA-DR antigen canbe used in treatment of both B-cell and T-cell disorders. Particularlypreferred B-cell antigens are those equivalent to human CD19, CD22,CD21, CD23, CD74, CD8O, and HLA-DR antigens. Particularly preferredT-cell antigens are those equivalent to human CD4, CD8 and CD25antigens. CD46 is an antigen on the surface of cancer cells that blockcomplement-dependent lysis (CDC). Preferred malignant melanomaassociated antigens are those equivalent to MART-1, TRP-1, TRP-2 andgp100. Further, preferred multiple myeloma-associated antigens are thoseequivalent to MUC1 and CD38.

Further, the present invention contemplates the administration of animmunoconjugate for diagnostic and therapeutic uses in B cell lymphomasand other disease or disorders. An immunoconjugate, as described herein,is a molecule comprising an antibody component and a therapeutic ordiagnostic agent, including a peptide that may bear the diagnostic ortherapeutic agent. An immunoconjugate retains the immunoreactivity ofthe antibody component, i.e., the antibody moiety has about the same orslightly reduced ability to bind the cognate antigen after conjugationas before conjugation.

A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Thetherapeutic agents recited here are those agents that also are usefulfor administration separately with the naked antibody as describedabove. Therapeutic agents include, for example, chemotherapeutic drugssuch as vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, COX-2 inhibitors,antimitotics, antiangiogenic and apoptotoic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others fromthese and other classes of anticancer agents, and the like. Other usefulcancer chemotherapeutic drugs for the preparation of immunoconjugatesand antibody fusion proteins include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, pyrimidine analogs, purine analogs, platinum coordinationcomplexes, hormones, and the like. Suitable chemotherapeutic agents aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated. For example, a therapeutically useful immunoconjugate can beobtained by conjugating a photoactive agent or dye to an antibodycomposite. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been termed photoradiation,phototherapy, or photodynamic therapy (Joni et al. (eds.), PHOTODYNAMICTHERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroff et. al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);idem., Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin.Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422(1989); Pelegrin et al., Cancer 67:2529 (1991). However, these earlierstudies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

Also contemplated by the present invention is the use of radioactive andnon-radioactive agents as diagnostic agents. A suitable non-radioactivediagnostic agent is a contrast agent suitable for magnetic resonanceimaging, computed tomography or ultrasound. Magnetic imaging agentsinclude, for example, non-radioactive metals, such as manganese, ironand gadolinium, complexed with metal-chelate combinations that include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, when used alongwith the antibodies of the invention. See U.S. Ser. No. 09/921,290 filedon Oct. 10, 2001, which is incorporated in its entirety by reference.

Furthermore, a radiolabeled antibody or immunoconjugate may comprise aγ-emitting radioisotope or a positron-emitter useful for diagnosticimaging. Suitable radioisotopes, particularly in the energy range of 60to 4,000 keV, include ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, ⁶²Cu, ⁶⁴Cu, ¹¹¹In, ⁶⁷Ga,⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, and the like. See,for example, U.S. Patent Application entitled “Labeling Targeting Agentswith Gallium-68”- Inventors G. L. Griffiths and W. J. McBride, (U.S.Provisional Application No. 60/342,104), which discloses positronemitters, such as ¹⁸F, ⁶⁸Ga, ^(94m)Tc. and the like, for imagingpurposes and which is incorporated in its entirety by reference.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an antibody fusion protein of ananti-CD74 antibody of the present invention. Other toxins suitablyemployed in the preparation of such conjugates or other fusion proteins,include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journalfor Clinicians 44:43 (1994). Additional toxins suitable for use in thepresent invention are known to those of skill in the art and aredisclosed in U.S. Pat. No. 6,077,499, which is incoporated in itsentirety by reference.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with the humanized anti-CD20 antibodies of the presentinvention. Suitable cytokines for the present invention include, but arenot limited to, interferons and interleukins, as described below.

Also contemplated by the present invention is a vaccine comprising thehumanized, chimeric or human CD74 mAabs or fragments thereof or anantibody fusion protein thereof covalently linked to Class-1 or Class-IIMHC antigenic peptides forming an antibody conjugate, wherein thevaccine is used to treat patients with cancer or infectious disease.When the antibody conjugate is internalized by the cell containing theCD74 marker, the Class-1 or Class-II antigenic peptides are released byprotolytic digestion from a larger peptide or protein linked to the Mabor fragment thereof, by a antigen presenting cell, such as a dendriticcell. This antibody conjugate is prepared by fusing cDNA coding for themAb or fragment thereof with cDNA coding for the antigenic peptide orprotein, and expressing the fusion protein in a bacteria, yeast, ormammalian cell. Antibody conjugates containing the humanized, chimericor human CD74 MAbs or fragments thereof or antibody fusion proteins ofthe present invention are particularly useful in a method of treatmentdescribed in pending U.S. Ser. No. 08/577,106, filed on Dec. 22, 1995,entitled “Use of Immunoconjugates to Enhance the Efficacy of Multi-StageCascade Boosting Vaccines,” which is herein incorporated in its entiretyby reference. The humanized anti-CD74 MAb of the present invention isparticularly useful in place of the murine LL 1 in Example 5.

Preparation of Immunoconjugates

Any of the antibodies or antibody fusion proteins of the presentinvention can be conjugated with one or more therapeutic or diagnosticagents. Generally, one therapeutic or diagnostic agent is attached toeach antibody or antibody fragment but more than one therapeutic agentor diagnostic agent can be attached to the same antibody or antibodyfragment. The antibody fusion proteins of the present invention comprisetwo or more antibodies or fragments thereof and each of the antibodiesthat comprises this fusion protein can contain a therapeutic agent ordiagnostic agent. Additionally, one or more of the antibodies of theantibody fusion protein can have more than one therapeutic of diagnosticagent attached. Further, the therapeutic agents do not need to be thesame but can be different therapeutic agents. For example, one canattach a drug and a radioisotope to the same fusion protein.Particulary, an IgG can be radiolabeled with ¹³¹I and attached to adrug. The ¹³¹I can be incorporated into the tyrosine of the IgG and thedrug attached to the epsilon amino group of the IgG Iysines. Boththerapeutic and diagnostic agents also can be attached to reduced SHgroups and to the carbohydrate side chains.

Bispecific antibodies of the present invention are useful inpretargeting methods and provide a preferred way to deliver twotherapeutic agents or two diagnostic agents to a subject. U.S. Ser. No.09/382,186 discloses a method of pretargeting using a bispecificantibody, in which the bispecific antibody is labeled with ¹²⁵I anddelivered to a subject, followed by a divalent peptide labeled with^(99m)Tc. The delivery results in excellent tumor/normal tissue ratiosfor ¹²⁵I and ^(99m)Tc, thus showing the utility of two diagnosticradioisotopes. Any combination of known therapeutic agents or diagnosticagents can be used to label the antibodies and antibody fusion proteins.The binding specificity of the antibody component of the mAb conjugate,the efficacy of the therapeutic agent or diagnostic agent and theeffector activity of the Fc portion of the antibody can be determined bystandard testing of the conjugates.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such peptides can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same peptide that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

The Fc region is absent if the antibody used as the antibody componentof the immunoconjugate is an antibody fragment. However, it is possibleto introduce a carbohydrate moiety into the light chain variable regionof a full-length antibody or antibody fragment. See, for example, Leunget al., J Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of whichare incorporated in their entirety by reference. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

Pharmaceutically Acceptable Excipients

The humanized, chimeric and human anti-CD74 mAbs to be delivered to asubject can consist of the mAb alone, immunoconjugate, fusion protein,or can comprise one or more pharmaceutically suitable excipients, one ormore additional ingredients, or some combination of these.

The immunoconjugate or naked antibody of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the immunoconjugate or naked antibody are combinedin a mixture with a pharmaceutically suitable excipient. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

The immunoconjugate or naked antibody of the present invention can beformulated for intravenous administration via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic conjugate or nakedantibody. Control release preparations can be prepared through the useof polymers to complex or absorb the immunoconjugate or naked antibody.For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate, antibody fusion proteins, or naked antibody mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. In general, the dosage of anadministered immunoconjugate, fusion protein or naked antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of immunoconjugate, antibody fusion protein or naked antibodythat is in the range of from about 1 mg/kg to 20 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

For purposes of therapy, the immunoconjugate, fusion protein, or nakedantibody is administered to a mammal in a therapeutically effectiveamount. A suitable subject for the present invention is usually a human,although a nonhuman animal subject is also contemplated. An antibodypreparation is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal. Inparticular, an antibody preparation of the present invention isphysiologically significant if its presence invokes an antitumorresponse or mitigates the signs and symptoms of an autoimmune diseasestate. A physiologically significant effect could also be the evocationof a humoral and/or cellular immune response in the recipient mammal.

Methods of Treatment

The present invention contemplates the use of naked anti-CD74 antibodiesof the present invention as the primary composition for treatment of aCD74 expressing malignancy, where the disease or disorder is selectedfrom the group consisting of an immune dysregulation disease, anautoimmune disease, organ graft rejection, and graft versus hostdisease. The CD74 expressing malignancy is selected from the groupconsisting of a solid tumor, non-Hodgkin's lymphoma, Hodgkin's lymphoma,multiple myeloma, another B-cell malignancy and a T-cell malignancy. Thesolid tumor is selected from the group consisting of a melanoma,carcinoma and sarcoma and the carcinoma is selected from the groupconsisting of a renal carcinoma, lung carcinoma, intestinal carcinoma,stomach carcinoma and melanoma. The B-cell malignany is selected fromthe group consisting of non-Hodgkins lymphoma, Hodkgin's lymphoma,indolent forms of B-cell lymphomas, aggressive forms of B-celllymphomas, chronic lymphatic leukemias, acute lymphatic leukemias, andmultiple myeloma, B-cell disorders and other diseases. In particular,the compositions described herein are particularly useful for treatmentof various autoimmune as well as indolent forms of B-cell lymphomas,aggressive forms of B-cell lymphomas, chronic lymphatic leukemias, acutelymphatic leukemias, multiple myeloma, and Waldenstrom'smacroglobulinemia. For example, the humanized anti-CD74 antibodycomponents and immuno conjugates can be used to treat both indolent andaggressive forms of non-Hodgkin's lymphoma.

More specifically, the invention contemplates a method for treating aB-cell malignancy comprising administering to a subject with a B-cellrelated malignancy, a therapeutic composition comprising apharmaceutically acceptable carrier and at least one humanized,chimeric, or human anti-CD74 mAb or fragment thereof or antibody fusionprotein thereof of the present invention, wherein the B-cell malignancyis lymphoma or leukemia. More specifically, the B-cell malignancy isnon-Hodgkin's lymphoma, indolent forms of B-cell lymphomas, aggressiveforms of B-cell lymphomas, multiple myeloma, chronic lymphaticleukemias, or acute lymphatic leukemias. The CD74 mAb or fragmentthereof is administered intravenously or intramuscularly at a dose of20-2000 mg. The present method further comprises administering theanti-CD74 mAb or fragment thereof before, during or after theadministration of at least one therapeutic agent used to treat theB-cell malignancy. The therapeutic agent comprises a naked antibody, animmunomodulator, a hormone, a cytotoxic agent, an enzyme, an antibodyconjugated to at least one immunomodulator, radioactive label, hormone,enzyme, or cytotoxic agent, or a combination thereof The immunomodulatorpreferably is a cytokine and said cytotoxic agent is a drug or toxin.The antibody that is administered incombination as a naked antibody oras a supplemental immunoconjugate is reactive with CD4, CD5, CD8, CD14,CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40,CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, Ia, HM1.24, tenascin,and HLA-DR, preferably a mature HLA-DR dimer, formulated in apharmaceutically acceptable vehicle.

The invention also contemplates treating a malignancy comprisingadministering to a subject with a CD74 antigen-positive malignancy otherthan lymphoma or leukemia, a therapeutic composition comprising apharmaceutically acceptable carrier and at least one anti-CD74 mAb orfragment thereof or an antibody fusion protein thereof as disclosed inthe present invention. The anti-CD74 mAb or fragment thereof or anantibody fusion protein thereof is administered intravenously orintramuscularly at a dose of 20-2000 mg. Further, the anti-CD74 mAb orfragment thereof or an antibody fusion protein thereof is administeredbefore, during or after the administration of at least one therapeuticagent used to treat the malignancy. The therapeutic agent, as describedabove and throughout the specification, comprises an antibody, animmunomodulator, a hormone, a cytotoxic agent, an antibody conjugated toat least one immunomodulator, radioactive label, enzyme, hormone,cytotoxic agent, antisense oligonucleotide, or a combination thereof,where the immunomodulator is a cytokine and said cytotoxic agent is adrug or toxin. When an antibody is administered in combination with theanti-CD74 mAb or fragment thereof to treat a malignancy that is not aB-cell malignancy, it should be reactive with a tumor marker other thanCD74, expressed by the cells that comprise the malignancy that istreated, formulated in a pharmaceutically acceptable vehicle. Examplesof antibodies that can be administered for malignant melanoma associatedantigens are those antibodies reactive with MART-1, TRP-1, TRP-2 andgp100. Further, preferred antibodies to multiple myeloma-associatedantigens are those reactive with MUC1 and CD38.

The compositions for treatment contain at least one humanized, chimericor human monoclonal anti-CD74 antibody alone or in combination withother antibodies, such as other humanized, chimeric, or humanantibodies, therapeutic agents or immunomodulators. In particular,combination therapy with a fully human antibody is also contemplated andis produced by the methods as set forth above.

Naked or conjugated antibodies to the same or different epitope orantigen may also be combined with one or more of the antibodies of thepresent invention. For example, a humanized, chimeric or human nakedanti-CD74 antibody may be combined with another naked humanized, nakedchimeric or naked human anti-CD74 mAb; a humanized, chimeric or humannaked anti-CD74 antibody may be combined with an anti-CD74immunoconjugate; a naked anti-CD74 antibody may be combined with ananti-CD22 radioconjugate; or an anti-CD22 naked antibody may be combinedwith a humanized, chimeric or human anti-CD74 antibody conjugated to anisotope, or one or more chemotherapeutic agents, cytokines, enzymes,toxins or a combination thereof. A fusion protein of a humanized,chimeric or human CD20 antibody and a toxin or immunomodulator, or afusion protein of at least two different B-cell antibodies (e.g., a CD74and a CD22 mAb, a CD20 mAb or a CD19 mAb) may also be used in thisinvention. Reference is made to pending U.S. Ser. No. 09/965,796 filedon Oct. 1, 2001, entitled “Immunotherapy of B-Cell Malignancies UsingAnti-CD-22 Antibodies,” which is a continuation of U.S. Pat. No.6,306,393, both of which are incorporated in their entirety byreference, that discloses treatment with an anti-CD22 antibodies incombination with other naked antibodies. Many different antibodycombinations, targeting at least two different antigens associated withB-cell disorders, as listed already above, may be constructed, either asnaked antibodies or as partly naked and partly conjugated with atherapeutic agent or immunomodulator, or merely in combination withanother therapeutic agents, such as a cytotoxic drug or with radiation.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, Iymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulatingfactors (e.g., granulocytecolony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferons(e.g., interferons-α, -β and -γ), the stem cell growth factor designated“S1 factor,” erythropoietin, thrombopoietin or a combination thereof.Examples of suitable immunomodulator moieties include IL-1, IL-2, IL-3,IL-6, IL-10, IL-12, IL-18, IL-21, and a combination thereof, andinterferon-γ, TNF-α, and the like. Alternatively, subjects can receivenaked anti-CD74 antibodies and a separately administered cytokine, whichcan be administered before, concurrently or after administration of thenaked anti-CD74 antibodies. As discussed supra, the anti-CD74 antibodymay also be conjugated to the immunomodulator. The immunomodulator mayalso be conjugated to a hybrid antibody consisting of one or moreantibodies binding to different antigens.

Multimodal therapies of the present invention further includeimmunotherapy with naked anti-CD74 antibodies supplemented withadministration of anti-CD22, anti-CD 19, anti-CD21, anti-CD20,anti-CD80, anti-CD23, anti-CD46 or HLA-DR, preferably the mature HLA-DRdimer antibodies in the form of naked antibodies, fusion proteins, or asimmunoconjugates. These antibodies include polyclonal, monoclonal,chimeric, human or humanized antibodies that recognize at least oneepitope on these antigenic determinants. Anti-CD19 and anti-CD22antibodies are known to those of skill in the art. See, for example,Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., CancerImmunol. Immunother. 32:364 (1991); Longo, Curro Opin. Oncol. 8:353(1996) and U.S. Pat. Nos. 5,798,554 and 6,187,287, incorporated in theirentirety by reference.

In another form of multimodal therapy, subjects receive naked anti-CD74antibodies, and/or immunoconjugates, in conjunction with standard cancerchemotherapy. For example, “CVB” (1.5 g/m² cyclophosphamide, 200-400mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen used totreat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate and brostatin-1. In a preferred multimodal therapy, bothchemotherapeutic drugs and cytokines are co-administered with anantibody, immunoconjugate or fusion protein according to the presentinvention. The cytokines, chemotherapeutic drugs and antibody orimmunoconjugate can be administered in any order, or together.

In a preferred embodiment, NHL is treated with 4 weekly infusions of thehumanized anti-CD74 antibody at a dose of 200-400 mg/m² weekly for 4consecutive weeks or every-other week (iv over 2-8 hours), repeated asneeded over next months/yrs. Also preferred, NHL is treated with 4semi-monthly infusions as above, but combined with epratuzumAb(anti-CD22 humanized antibody) on the same days, at a dose of 360mg,/m², given as an iv infusion over 1 hour, either before, during orafter the anti-CD74 monoclonal antibody infusion. Still preferred, NHLis treated with 4 weekly infusions of the anti-CD74 antibody as above,combined with one or more injections of CD22 mAb radiolabeled with atherapeutic isotope such as yttrium-90 (at dose of Y⁹⁰ between 5 and 35mCi/meter-square as one or more injections over a period of weeks ormonths.

In addition, a therapeutic composition of the present invention cancontain a mixture or hybrid molecules of monoclonal naked anti-CD74antibodies directed to different, non-blocking CD74 epitopes.Accordingly, the present invention contemplates therapeutic compositionscomprising a mixture of monoclonal anti-CD74 antibodies that bind atleast two CD74 epitopes. Additionally, the therapeutic compositiondescribed herein may contain a mixture of anti-CD74 antibodies withvarying CDR sequences.

Although naked anti-CD74 antibodies are the primary therapeuticcompositions for treatment of B cell lymphoma and autoimmune diseases,the efficacy of such antibody therapy can be enhanced by supplementingthe naked antibodies, with supplemental agents, such asimmunomodulators, like interferons, including IFNα, IFNβ and IFNγ,interleukins including IL-1, IL-2, IL-3, IL-6, IL-10, IL12, IL-15,IL-18, IL-21, and a combination thereof, and cytokines including G-CSFand GM-CSF. Accordingly, the CD74 antibodies can be combined not onlywith antibodies and cytokines, either as mixtures (given separately orin some predetermined dosing regiment) or as conjugates or fusionproteins to the anti-CD74 antibody, but also can be given as acombination with drugs or with antisense olignucleotides. For example,the anti-CD74 antibody may be combined with CHOP as a 4-drugchemotherapy regimen. Additionally, a naked anti-CD74 antibody may becombined with a naked anti-CD22 antibodies and CHOP or Fludarabine as adrug combination for NHL therapy. The supplemental therapeuticcompositions can be administered before, concurrently or afteradministration of the anti-CD74 antibodies. The naked anti-CD74 mAb mayalso be combined with an antisense bel oligonucleotide.

As discussed supra, the antibodies of the present invention can be usedfor treating B cell lymphoma and leukemia, and other B cell diseases ordisorders as well as other malignancies in which affected or associatedmalignant cells are reactive with CD74. For example, anti-CD74antibodies can be used to treat immune dysregulation disease and relatedautoimmune diseases, including Class-III autoimmune diseases such asimmune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpura and chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sjögren's syndrome, multiple sclerosis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, sarcoidosis, ulcerativecolitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa,ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.

Particularly, the humanized, chimeric or human anti-CD74 mAbs orfragments thereof or antibody fusion proteins thereof of the presentinvention are administered to a subject with one or more of theseautoimmune diseases. The anti-CD74 antibodies of the present inventionare particularly useful in the method of treating autoimmune disorders,disclosed in pending U.S. Ser. No. 09/590,284 filed on Jun. 9, 2000entitled “Immunotherapy of Autoimmune Disorders using Antibodies thatTarget B-Cells,” which is incorporated in its entirety by reference.Preferably the anti-CD74 mAb or fragment thereof or an antibody fusionprotein thereof is administered intravenously or intramuscularly at adose of 20-2000 mg. Further, the anti-CD74 mAb or fragment thereof or anantibody fusion protein thereof is administered before, during or afterthe administration of at least one therapeutic agent used to treat thedisorder. The therapeutic agent, as described above and throughout thespecification, comprises an antibody, an immunomodulator, a hormone, anenzyme, a cytotoxic agent, an antibody conjugated to at least oneimmunomodulator, radioactive label, hormone, enzyme, or cytotoxic agent,antisense oligonucleotide or a combination thereof, where theimmunomodulator is a cytokine and said cytotoxic agent is a drug ortoxin. The antibody that is administered incombination as a nakedantibody or as a supplemental immunoconjugate is reactive with CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD37,CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, Ia, HM1.24,tenascin, and mature HLA-DR, preferably a mature HLA-DR dimer,formulated in a pharmaceutically acceptable vehicle.

101211 A further method for treating one of the diseases selected fromthe group consisting of lymphoma, leukemia, myeloma, otherCD74-expressing malignancies, immune dysregulation disease, autoimmunedisease and a combination thereof, comprising administering atherapeutic composition comprising a pharmaceutically acceptable carrierand at least one anti-CD74 mAb or fragment thereof or an antibody fusionprotein thereof of the present invention, wherein at least onetherapeutic agent is linked to the mAb or fragment thereof or the Fvs orFab's of the antibody fusion protein thereof by chemical conjugation orby genetic fusion. The therapeutic agent may be an immunomodulator, aradioactive label, a hormone, an enzyme, or a cytotoxic agent, and theimmunomodulator is a cytokine and said cytotoxic agent is a drug ortoxin.

Anti-CD74 antibodies may also induce apoptosis in cells expressing theCD74 antigen. Evidence of this induction is supported in the examples ofthe present invention. Other antibodies have demonstrated that apoptosiscould be induced using lymphoid cells that have Fc-receptors reactivewith the IgG 1-Fc of CD20 MAbs that crosslinked. See Shan et al., CancerImmunol. Immunother. 48(12):673-683 (2000). Further, it was reportedthat aggregates of a chimeric CD20 MAb, i.e., homopolymers, inducedapoptosis. See Ghetie et al., Blood 97(5): 1392-1398 (2000) and Ghetieet al., Proc. Natl. Acad. Sci USA 94(14): 7509-7514 (1997).

Antibodies specific to the CD74 surface antigen of B cells can beinjected into a mammalian subject, which then bind to the CD74 cellsurface antigen of both normal and malignant B cells. A mammaliansubject includes humans and domestic animals, including pets, such asdogs and cats. The anti-CD74 mAbs of the present invention, i.e.,humanized, chimeric, human, caninized and felinized, and even murineanti-CD74 mAbs, can be used to treat the nonhuman mammalian subjectswhen there is a species cross reactivity for the CD74 antigen. Themurine mAbs, which are immunogenic in humans, are usually lessimmunogenic in non-human mammalian subjects. The anti-CD74 antibodybound to the CD74 surface antigen leads to the destruction and depletionof neoplastic B cells.

Method of /Diagnosis

Also provided for in the present invention is a method of diagnosing adisease in a subject, diagnosed with or suspected of having at least oneof the diseases selected from the groups consisting of lymphoma,leukemia, myeloma, other CD74-expressing malignancies, immunedysregulation disease, autoimmune disease and a combination thereof,comprising administering to said subject a diagnostically effectiveamount of a diagnostic conjugate a pharmaceutically acceptable carrierand at least one anti-CD74 mAb or fragment thereof or antibody fusionprotein thereof, wherein a diagnostic agent is linked to the mAb orfragment thereof or the Fvs or Fabs of the antibody fusion proteinthereof by chemical conjugation and detecting the diagnostic agent.Diagnostic agents useful in the present invention are a radioisotope,wherein the photons of the radioisotope are detected by radioscintigrapyor PET, or a metal that can be detected by MRI, or a liposome or gasfilled liposome, and wherein the liposome can be detected by anultrasound scanning device.

The internalization of murine anti-CD74 mAb, chimeric anti-CD74 mAb andhumanized anti-CD74 mAb into target cells can be followed byfluorescence labeling, essentially according to the procedure of Pirkeret al., J. Clin. Invest., 76:1261 (1985), which is incorporated byreference. Cultured Raji cells are centrifuged and the cells resuspendedin fresh medium to a concentration of about 5×10⁶ cells/ml. To each wellof a 96-well microtiter plate, 100 μl of the cell suspension is added.The antibodies, 40 μg/ml, in a volume of 100 μl are added to thereaction wells at timed intervals so as to terminate all reactionssimultaneously. The plate is incubated at 37° C. in a C0₂ cell cultureincubator. Unbound antibodies are removed by washing the cells threetimes with cold 1% FCS/PBS at the end of the incubation. The cells arethen treated with 1 ml of Formaid-Fresh [10% formalin solution (Fisher,Fair Lawn, N.J.)] for 15 min at 4° C. After washing, antibodies presenteither on the cell surface or inside the cells are detected by treatmentwith FITC-labeled goat anti-mouse antibody (Tago, Burlingame, Calif.),or FITC-labeled goat anti-human antibody (Jackson ImmunoResearch, WestGrove, Pa.), depending on whether the antibody being assayed for ismurine, chimeric, or humanized, respectively. Fluorescence distributionsare evaluated using a BH-2 fluorescence microscope (Olympus, LakeSuccess, N.Y.).

In a related vein, a method for screening/diagnosing bone cancers isdescribed in Juweid et al., 1999, could benefit from the superioranti-CD74 mAbs of the present invention. Accordingly, a methodcomprising ^(99m)Tc-labeled humanized or chimeric anti-CD74 mAb iscontemplated.

Expression Vectors

The DNA sequence encoding a humanized, chimeric or human anti-CD74 mAb.More specifically the DNA sequence comprises a nucleic acid encoding amAb or fragment thereof selected from the group consisting (a) ananti-CD74mAb or fragment described herein, (b) an immunoconjugatecomprising anyone of the anti-CD74 mAbs or fragment thereof describedherein, (c) an antibody fusion protein or fragment thereof comprising atleast two of said anti-CD74 mAbs or fragments thereof described herein;(d) an antibody fusion protein or fragment described herein; (e) avaccine as described herein; and a bispecific or multispecifc antibodydescribed herein. Any of the DNA sequences of the present invention canbe recombinantly engineered into a variety of known host vectors thatprovide for replication of the nucleic acid. These vectors can bedesigned, using known methods, to contain the elements necessary fordirecting transcription, translation, or both, of the nucleic acid in acell to which it is delivered. Known methodology can be used to generateexpression constructs the have a protein-coding sequence operably linkedwith appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques and synthetictechniques. For example, see Sambrook et al., 1989) MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory (New York); Ausubel etal., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons(New York). Also provided for in this invention is the delivery of apolynucleotide not associated with a vector.

Vectors suitable for use in the instant invention can be viral ornon-viral. Particular examples of viral vectors include adenovirus, AAV, herpes simplex virus, lentivirus, and retrovirus vectors. An exampleof a non-viral vector is a plasmid. In a preferred embodiment, thevector is a plasmid.

An expression vector, as described herein, is a polynucleotidecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements. Preferred expression vectors are thepdHI2 and GS vector.

Preferably, the expression vector of the instant invention comprises theDNA sequence encoding a humanized, chimeric or human anti-CD74 mAb,which includes both the heavy and the light chain variable and constantregions. However, two expression vectors may be used, with onecomprising the heavy chain variable and constant regions and the othercomprising the light chain variable and constant regions. Stillpreferred, the expression vector further comprises a promoter. Becauseany strong promoter can be used, a DNA sequence encoding a secretionsignal peptide, a genomic sequence encoding a human IgG1 heavy chainconstant region, an Ig enhancer element and at least one DNA sequenceencoding a selection marker.

The method for the expression of an anti-CD74mAb or fragment thereof orantibody fusion protein or fragment thereof employing the presentinvention comprises: (a) transfecting a host cell with a DNA sequenceencoding an anti-CD74 mAb or fragment thereof or an immunoconjugate,fusion protein or bispecific or multispecific antibody thereof; and (b)culturing the cell secreting the antiCD74 mAb or fragment thereof orantibody fusion protein or fragment thereof. The host cell is derivedfrom bacterial, yeast or mammalian cells. More preferably from amammalian cells, which in one embodiment is a Iymphocyctic cell, such asa myeloma cell.

Also contemplated herein is a method for expressing a humanized anti-CD74 mAb, comprising (i) linearizing at least one expression vectorcomprising a DNA sequence encoding a humanized, chimeric, or humananti-CD74 mAb, (ii) transfecting mammalian cells with at least one ofsaid linearized vector, (iii) selecting transfected cells which expressa marker gene, and (iv) identifying the cells secreting the humanizedanti-CD74 mAb from the transfected cells.

The inventors have isolated cDNAs encoding the VL and VH regions of themurine anti-CD74 monoclonal antibody (mLL1 mAb) and recombinantlysubcloned them into mammalian expression vectors containing the genesencoding kappa and IgG1 constant regions, respectively, of humanantibodies. Cotransfection of mammalian cells with these two recombinantDNAs expressed a chimeric antiCD74 mAb (cLL1) that, like the parent mLL1mAb, bound avidly to, and was rapidly internalized by, B-Iymphoma cells.

The CDRs of the VK and VH DNAs have been similarly recombinantly linkedto the framework (FR) sequences of the human VK and VH regions,respectively, which are subsequently linked, respectively, to the humankappa and IgG1 constant regions, so as to express in mammalian cells asdescribed above a humanized anti-CD74 mAb (hLL1).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

The antibody described herein is a monoclonal antibody (mAb). Monoclonalantibodies are a homogeneous population of antibodies to a particularantigen and the antibody comprises only one type of antigen binding siteto which the nucleic acid specifically binds. Rodent monoclonalantibodies to specific antigens may be obtained by methods known tothose skilled in the art. See, for example, Kohler and Milstein, Nature256: 495 (1975), and Coligan et al. (eds.), CURRENT PROOCOLS INIMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[hereinafter “Coligan”]. Briefly, monoclonal antibodies can be obtainedby injecting mice with a composition comprising an antigen, verifyingthe presence of antibody production by removing a serum sample, removingthe spleen to obtain B-lymphocytes, fusing the B-lymphocytes withmyeloma cells to produce hybridomas, cloning the hybridomas, selectingpositive clones which produce antibodies to the antigen, culturing theclones that produce antibodies to the antigen, and isolating theantibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL 10, pages 79-104 (The Humana Press, Inc. 1992).

Method of Making

The VK and VH sequences for chimeric or humanized anti-CD74 mAb canamplified by PCR as described by Orlandi et al., (Proc. Natl. Acad.Sci., USA, 86: 3833 (1989)) which is incorporated by reference. VKsequences may be amplified using the primers CK3BH and VK5-3 (Leung etal., BioTechniques, 15: 286 (1993), which is incorporated by reference),while VH sequences can be amplified using the primer CH1B which annealsto the CH1 region of murine IgG, and VHIBACK (Orlandi et al., 1989above). The PCR reaction mixtures containing 10 μl of the first strandcDNA product, 9 μl of 10× PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH8.3), 15 mM MgCl2, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus,Norwalk, Conn.), can be subjected to 30 cycles of PCR. Each PCR cyclepreferably consists of denaturation at 94° C. for 1 min, annealing at50° C. for 1.5 min, and polymerization at 72° C. for 1.5 min. AmplifiedVK and VH fragments can be purified on 2% agarose (BioRad, Richmond,Calif.). See Example 3 for a method for the synthesis of an oligo A(149-mer) and an oligo B (140-mer) on an automated Cyclone Plus DNAsynthesizer (Milligan-Biosearch) for use in constructing humanized Vgenes.

PCR products for VK can be subcloned into a staging vector, such as apBR327-based staging vector VKpBR that contains an Ig promoter, a signalpeptide sequence and convenient restriction sites to facilitate in-frameligation of the VK PCR products. PCR products for VH can be subclonedinto a similar staging vector, such as the pBluescript-based VHpBS.Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al., Proc. Natl.Acad. Sci., USA, 74: 5463 (1977) which is incorporated by reference.

The DNA sequences described herein are to be taken as including allalleles, mutants and variants thereof, whether occurring naturally orinduced.

The two plasmids can be co-transfected into an appropriate cell, e.g.,myeloma Sp2/0-Ag 14, colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of chimeric or humanizedanti-CD74 mAbs by, for example, an ELISA assay, as described below.

Transfection, and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of hLL1 pith (light chain expressionvector) and 20 μg of hLL1pG1g(heavy chain expression vector) can be usedfor the transfection of 5×10⁶ SP2/0 myeloma cells by electroporation(BioRad, Richmond, Calif.) according to Co et al., J. Immunol., 148:1149 (1992) which is incorporated by reference. Following transfection,cells may be grown in 96-well microtiter plates in complete HSFM medium(GIBCO, Gaithersburg, MD) at 37° C., 5% CO₂. The selection process canbe initiated after two days by the addition of hygromycin selectionmedium (Calbiochem, San Diego, Calif.) at a final concentration of 500μg/ml of hygromycin. Colonies typically emerge 2-3 weekspost-electroporation. The cultures can then be expanded for furtheranalysis.

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch, West Grove, Pa.) are added to the wells, (100 μl ofantibody stock diluted×10⁴, supplemented with the unconjugated antibodyto a final concentration of 1.0 μl/ml). Following an incubation of 1 h,the plates are washed, typically three times. A reaction solution, [100μl, containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbancy at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA, as before, and its concentration adjusted to about1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is conveniently added tothe sample as preservative.

All published articles and patents, as well as filings of patents citedherein are incorporated in their entirety by reference. The invention isfurther described by reference to the following examples, which areprovided for illustration only. The invention is not limited to theexamples but rather includes all variations that are evident from theteachings provided herein.

Examples Example 1 Molecular Cloning and Sequence Elucidation for LL1Heavy and Light Chain Variable Regions

The V gene of mLL1 was obtained by RT-PCR using VK5′-4 and VK1FORprimers as described by Leung et al. 1993 and Orlandi et al. (PNAS86:3833-3837. (1989), respectively, and cloned into pCR2.1 AT-cloningvector (Invitrogen). Multiple clones were sequenced to eliminatepossible errors resulted from PCR reaction. Majority of clones (6)contained an identical murine V sequence, which was designated as LL1Vand the sequence is shown in FIG. 1B. Comparison with other mouse Vksequences revealed LL1Vk is a member of the kappa light chain subclassII.

Since RT-PCR failed to yield a full-length sequence encoding a mouse VHgene, the second cloning approach, rapid amplification of cDNA 5′-ends(5′-RACE) was employed. The adaptor-ligated cDNA prepared from LL1hybridoma cells was amplified by PCR using a universal anchor primer(Life Technologies) and a gene specific primer, CH-1B (Leung et al.1994), which anneals to the CH1 region of murine heavy chain. The majorPCR species of ˜650 by resulted from PCR was cloned into pCR2.1AT-cloning vector and multiple clones were sequenced by DNA sequencing.The PCR product contained a full-length VH sequence (FIG. 1A) flanked bythe sequences of non-coding and secretion signal peptide at ‘5-end andpartial coding sequence for the CH1 domain of 1 chain. No defectivemutation was found within the sequence encoding the VH, which wasdesignated as LL1VH. Comparison of hLL1VH with other mouse VH sequencesrevealed that it belonged to mouse heavy chain subgroup miscellaneous(Kabat et al., 1991). By comparing the amino acid sequences of LL1VH andV with murine Ab V genes in Kabat database and following Kabat'sdefinition, the CDR regions of hLL1VH and Vk were identified as shown inFIGS. 1A and B, respectively. By comparing the amino acid sequences ofLL1VH and V with murine Ab V genes in Kabat database and followingKabat's definition, the CDR regions of hLL1VH and Vk were identified asshown in FIGS. 1A and B, respectively.

Example 2 Construction of the Expression Vector for Chimeric LL1

To evaluated the authenticity of the cloned Fv for LL1, a chimeric LL1(cLL1) was constructed and expressed. The nucleotide residues 7-12 ofLL1Vk were modified to a PvuII restriction site, CAGCTG, by PCR withprimers LL1VK-PvuII and VK1 FOR. The resulting PCR product was digestedwith PvuII and BglII (partially, due to the presence of an internalBglII site in the Vk) and force-cloned into a pBR327-based stagingvector (digested with PvuII and BclI), VKpBR2, which contained same Igpromoter, signal peptide sequence and convenient restriction sites tofacilitate in-frame ligation of the VK PCR product as used by Orlandi etal., 1989 and Leung et al., 1994.

(SEQ ID NO: 26) LL1VK-PvuII 5′GAT GTT CAG CTG ACC CAA ACT CCA CTC TCC-3′

Similarly, the nucleotide sequences at positions 10-15 and 345-351 ofLL1VH were converted to PstI and BstEII, respectively, by PCR withprimers LL1 B-1 and LL1F-1. The VH PCR product was then digested withPstI and BstEII and ligated into PstI and BstEII digested VHpBS2, apBluescript-based staging vector containing a signal peptide sequenceand convenient restriction sites to facilitate in-frame ligation of theVH PCR product {Orlandi, Gussow, et al., 1989 741/id}, modified fromVHpBS (Leung, S. O., Shevitz, J., Pellegrini, M. C., Dion, A. S., Shih,L. B., Goldenberg, D. M., and Hansen, H. J. (1994)).

(SEQ ID NO: 27) LL1B-1 5′-CAG ATC CAG CTG CAG CAG TCT GGA CCT GAG-3′(SEQ ID NO: 28) LL1F-1 5′-GA GAC GGT GAC CAG AGT CCC TTG GCC CCA A-3′

The sequences of both cLL1VH and Vk were confirmed by DNA sequencing andshown in FIGS. 2A and 2B, respectively.

The fragment containing the Vk sequences of cLL1, together with thesignal peptide sequences, were excised from LL1VKpBR2 by doublerestriction digestion with XbaI and BamHI. The ˜550 by Vk fragments wasthen subcloned into the XbaI/BamHI site of a mammalian expressionvector, pdHL2. The resulting vector designated as cLL1VkpdHL2.Similarly, the ca. 750 by fragments containing the LL1VH, together withthe signal peptide sequences, were excised from LL1VHpBS2 by XhoI andBamHI digestion and isolated by electrophoresis in an agarose gel. Thefragment was subcloned into the XhoI and HindIII site of cLL1VkpdHL2with the aid of linker comparable to both BamHI and HindIII ends,resulting in the final expression vectors, designated as cLL1pdHL2.

Example 3 Transfection and Expression of cLL1

Approximately 30 μg of cLL1pdHL2 was linerized by digestion with SaIland transfected into Sp2/0-Ag14 cells by electroporation. Thetransfected cells were plated into 96-well plate for 2 days and thenselected for MTX resistance. Supernatants from colonies survivingselection were monitored for chimeric antibody secretion by ELISA assay.Positive cell clones were expanded and cLL1 was purified from cellculture supernatant by affinity chromatograpgy on a Protein A column.

Example 4 Binding Activity Assays

A competition cell binding assay was carried out to assess theimmunoreactivity of cLL1 relative to the parent mLL1. A constant amountof ¹²⁵I-labeled mLL1 (100,000 cpm) was incubated with Raji cells in thepresence of varying concentrations of cLL1 or mLL1 at 4° C. for 1-2 h.The radioactivity associated with cells was determined after washing. Asshown in FIG. 5, cLL1 antibody exhibited comparable binding activity asthat of mLL1, confirming the authenticity of the cloned V genes.

The results were confirmed by a second competition assay based on flowcytometry. Briefly, using Raji cells as before and varying theconcentration of one antibody relative to other, as before, the amountof bound mLL1 or cLL1 was determined with FITC-labeled anti-mouse Fc oranti-human Fc antibodies followed by analysis using flow cytometry.

An ELISA competitive binding assay were carried out in Raji cellmembrane coated plate to assess the immunoreactivity of cLL1 relative tothe parent mLL1. Raji cell membrane fraction was prepared by sonicationand centrifugation. The crude membrane extracts were coated in 96-wellflat bottomed PVC plate by centrifugation and fixed with 0.1%glutaraldehyde. Constant amount of the biotinylated mLL1 mixed withvaying concentrations of mLL1 or cLL1 was added to the membrane coatedwells and incubated at room temperature for 1-2 h. After washing,HRP-conjugated streptavidin was added and incubated for 1 h at roomtemperature. The amount of HRP-conjugated streptavidin bound to themembrane-bound biotinylated mLL1 was revealed by reading A490 nm afterthe addition of a substrate solution containing 4 mMortho-phenylenediamine dihydrochloride and 0.04% H₂0₂.

Example 5 Choice of Human Frameworks and Sequence Design for theHumanization of LL1 Monoclonal Antibody

By comparing the variable (V) region framework (FR) sequences of cLL1 tothat of human antibodies in the Kabat data base, the FRs of cLL1 VH andVk were found to exhibit the highest degree of sequence homology to thatof the human antibodies, RF-TS3 VH and HF-21/28 Vk, respectively. Theamino acid sequences of are provided in FIGS. 3A and 3B and are comparedwith the cLL1VH and Vk sequences. Therefore, the FRs of RF-TS3 VH andthe HF-21/28 Vk and FRs were selected as the human frameworks onto whichthe CDRs for LL1 VH and Vk were grafted, respectively. The FR4 sequenceof NEWM, however, rather than that of RF-TS3, was used to replace theRF-TS3 FR4 sequence for the humanization of LL1 heavy chain. See FIG.3A. A few amino acid residues in the LL1 FRs that are close to theputative CDRs were maintained in hLL1 based on the guideline describedpreviously (Qu et aL, Clin. Cancer Rec. 5:3095s-3100s (1990)). Theseresidues are L46, F87 and Q100 of VK (FIG. 3B) and I36, K37, Q46, A68,F91 and S93 of VH (FIG. 3A). FIGS. 3A and 3B compare the human, chimericand humanized VH and Vk amino acid sequences. The dots indicate theresidues in the cLL1 and hLL1 that are identical to the correspondingresidues in the human VH and Vk sequences. The DNA and amino acidsequences of hLL1 VH and Vk are shown in FIG. 4A and 48, respectively.

Example 6 PCR/Gene Synthesis of the Humanized V Genes

A modified strategy as described by Leung et al. (Leung et al, 1994) wasused to construct the designed VK and VH genes for hLL1 using acombination of long oligonucleotide synthesis and PCR as illustrated inFIG. 5. For the construction of the hLL1 VH domain, two longoligonucleotides, hLL1VHA (176 mer) and hLL1VHB (165-mer) weresynthesized on an automated DNA synthesizer (Applied Biosystem). ThehLL1VHA sequence represents nt 20 to 195 of the hLL1VH domain:

(SEQ ID NO: 29) 5′-GGTCTGAGTT GAAGAAGCCT GGGGCCTCAG TGAAGGTTTCCTGCAAGGCT TCTGGATACA CCTTCACTAA CTATGGAGTG AACTGGATAA AGCAGGCCCCTGGACAAGGG CTTCAGTGGA TGGGCTGGAT AAACCCCAAC ACTGGAGAGC CAACATTTGATGATGACTTC AAGGGA-3′

The hLL1 VHB sequence represents the minus strand of the hLL1 VH domaincomplementary to nt 173 to 337:

(SEQ ID NO: 30) 5′-TCCCTTGGCC CCAATAAGCA AACCAGGCTT CGTTTTTACCCCTCGATCTT GAACAGAAAT ACACGGCAGT GTCGTCAGCC TTTAGGCTGC TGATCTGGAGATATGCCGTG CTGACAGAGG TGTCCAAGGA GAAGGCAAAT CGTCCCTTGA AGTCATCATCAAATG-3′

The 3′-terminal sequences (22 nt residues) of hLL1 VHA and B arecomplementory to each other. Under defined PCR condition, 3′-ends ofhLL1 VHA and B anneal to form a short double stranded DNA flanked by therest of the long oligonucleotides. Each annealed end serves as a primerfor the transcription of the single stranded DNA, resulting in a doublestrand DNA composed of the nt 20 to 337 of hLL1VH. This DNA was furtheramplified in the presence of two short oligonucleotides, hLL1VHBACK andhLL1VHFOR to form the full-length hLL1VH.

(SEQ ID NO: 31) hLL1VHBACK 5′-GTG GTG CTG CAG CAA TCT GGG TCT GAG TTCAAG AAG CT-3′ (SEQ ID NO: 32) hLL1VHFOR 5′-AAG TGG ATC CTA TAA TCA TTCCTA GGA TTA ATG-3′.

Minimum amount of hLL1VHA and B (determined empirically) was amplifiedin the presence of 10 μl of 10× PCR Buffer (500 mM KCl, 100 mM Tris-HClbuffer, pH 8.3, 15 mM MgCl₂), 2 mol of hLL1VHBACK and hLL1VHFOR, and 2.5units of Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Conn.). Thisreaction mixture was subjected to 3 cycles of PCR reaction consisting ofdenaturation at 94° C. for 1 minute, annealing at 45° C. for 1 minute,and polymerization at 72° C. for 1.5 minutes, and followed by 27 cyclesof PCR reaction consisting of denaturation at 94° C. for 1 minute,annealing at 55° C. for 1 minute, and polymerization at 72° C. for 1minute. Double-stranded PCR-amplified product for hLL1VH wasgel-purified, restriction-digested with Pstl and BstEII and cloned intothe complementary Pstl/BstEII sites of the heavy chain staging vector,VHpBS2.

For constructing the full length DNA of the humanized VK sequence,hLL1VKA (159-mer) and hLL1VKB (169-mer) were synthesized as describedabove. hLL1VKA and B were amplified by two short oligonucleotideshLL1VKBACK and hLL1VKFOR as described above.

The hLL1 VHA sequence represents nt 16 to 174 of the hLL1VH domain.

(SEQ ID NO: 33) 5′-CAGTCTCCAC TCTCCCTGCC CGTCACCCTT GGACAGCCGGCCTCCATCTC CTGCAGATCA AGTCAGAGCC TTGTACACAG AAATGGAAAC ACCTATTTACATTGGTTTCA GCAGAGGCCA GGCCAATCTC CAAGGCTCCT GATCTACACA GTTTCCAAC-3′

The hLL1VHB sequence represents the minus strand of the hLL1VH domaincomplementary to nt 153 to 321.

(SEQ ID NO: 34) 5′-TGTCCCAGCA CCGAACGTGG GAGGAACATG TGAACTTTGAGAGCAGAAAT AAACCCCAAC ATCCTCAGCC TCCACCCTGC TGATTTTCAG TGTGAAATCAGTGCCTGACC CACTGCCGCT GAATCTGTCT GGGACCCCAG AAAATCGGTT GGAAACTGTGTAGATCAGG-3′. (SEQ ID NO: 35) hLL1VKBACK 5′-GAT GTT CAG CTG ACT CAG TCTCCA CTC TCC CTG-3′ (SE ID NO: 36) hLL1VKFOR 5′-G TTA GAT CTC CAG TCG TGTCCC AGC ACC GAA CG-3′.

Gel-purified PCR products for hLL1Vk were restriction-digested withPvuII and BglIII and cloned into the complementary PvuI/BclI sites ofthe light chain staging vector, VKpBR2. The final expression vectorhLL1pdHL2 was constructed by sequencially subcloning the Xbal-BamHI andXhoI/BamHI fragments of hLL1Vk and VH, respectively, into pdHL2 asdescribed above.

Example 7 Transfection, Expression and Binding Activity Assays for hLL1

The methods for expression and binding activity assays for hLL1 weresame as described for cLL1.

An ELISA competitive binding assay using Raji cell membrane extractcoated plate was developed to assess the immunoreactivity of hLL1. Rajicell membrane fraction was prepared by sonication and centrifugation.The crude membrane extracts were coated in 96-well flat bottomed PVCplate by centrifugation and fixed with 0.1% glutaraldehyde. Constantamount of the biotinylated mLL1 mixed with varying concentrations ofmLL1 or cLL1 was added to the membrane coated wells and incubated atroom temperature for 1-2 h. After washing, HRP-conjugated streptavidinwas added and incubated for 1 h at room temperature. The amount ofHRP-conjugated streptavidin bound to the membrane bound biotinylatedmLL1 was revealed by reading A_(490 nm) after the addition of asubstrate solution containing 4 mM ortho-phenylenediaminedihydrochloride and 0.04% H₂0₂. As shown by the competition assays inFIG. 6, mLL1 and cLL1 antibodies exhibited similar binding activities.Likewise, the competition assays in FIG. 7, hLL1 and cLL1 antibodiesexhibited similar binding activities.

Example 8 Internalization of hLL1

Standard antibody processing assay was used to evaluate theinternalization and metabolism of hLL1 in Raji cells (Hansen et al.,1996). Cells (10⁷) were incubated in 1 ml of tissue culture mediumcontaining ¹²⁵I-labeled hLL1 or LL1 (10⁷ cpm) for 1 h at 37° C. Toensure the specificity of Ab binding, controls of 1/10 sample size(cells, radioactivity and medium) were set up in every experiment withand without excess unlabeled Ab (a final concentration of 100 μg/ml).After the binding incubation, unbound radioactivity was removed bywashing. The specificity controls were counted. In all experiments, thebinding of radioactivity to cells was at least 90% blocked by theunlabeled Ab. The cells were then resuspended in 30 ml of fresh mediumand dispensed in a 24-well plate with 1.5 ml/well. Samples of 1.5 mlwere saved for radioactivity determination, which was the initiallybound cpm. The plate was incubated in a CO₂ incubator. At 3, 24, 48, and72 h, the cells were collected as follows. Cells were resuspended byrepeated pipetting and transferred to conical tubes. The wells andpipette were rinsed with 1 ml fresh culture medium, which was added tothe initial cell suspension collected. The tube was centrifuged for 10min at 600×g and 1 ml of supernatant was carefully collected (40% of thetotal supernatant) and counted for radioactivity. BSA was added ascarrier protein to a final concentration of 1% and the protein wasprecipitated with 5 ml of cold 10% (w/v) trichloroacetic acid (TCA).After incubation for 30 min at 4° C. and centrifugation for 15 min at5000×g, the supernatant was discarded and the precipitated protein wascounted for radioactivity. The radiolabeled protein that was notprecipitated by TCA was considered degraded, and precipitatedradioactive protein was considered intact. The cell pellet was countedfor the radioactivity remaining in the cells after being washed.Radioactivity in each fraction was expressed as a percentage of thatinitially bound. As shown in FIG. 8A, hLL1 showed similar rapidinternalizing and catabolic manner as murine LL1 after bound to thesurface of Raji cells, i.e. almost all of the bound radioactivity wascatabolized and released into the supernatant within 3 h. This is muchfaster than with other internalizing Abs, such as anti-CD22 andanti-CD19 (Hansen et al., 1996). The studies with early time pointsconfirmed the similar processing patterns of hLL1 and mLL1. Mostcatabolism was accomplished within one hour (FIG. 8B).

Example 9 Cytotoxicity of hLL1

The cytotoxic effect of hLL1 was compared with that of mLL1 and cLL1 inRaji cells, a human lymphoma cell line. Goat anti-human IgG Fc fragmentspecific Ab (α-hFc) was used as the crosslinker for hLL1 and cLL1 andgoat antimouse IgG Fc specific Ab (α-mFc) was used for mLL1. 5×10⁵ Rajicells were seeded at day 0 in 1 ml of culture medium containing 5 μg/mlof a LL1 Ab and 50 μg/ml of the appropriate crosslinker. The numbers oftotal and viable cells were counted daily for 3 days. As shown in FIG.9, The total number of normal Raji cells increased 4-5 fold in 3 daysand cell viability remained >80% at the end of third day. Cells treatedwith a crosslinker alone, a LL1 Ab alone, or a LL1 Ab with anuncomparable crosslinker (e.g. hLL1 and goat anti-mouse IgG Fc specificAb), were indistinguishable from normal Raji cells. However, acombination of hLL1 and anti-human IgG Fc specific Ab effectively causedcell death: >40% reduction in cell viability in one day and almost totalcell death in 3 days. The effectiveness of hLL1 was comparable with thatof mLL1 and cLL1. Similar results were observed when Daudi cells wereused (FIG. 10). No such effect was observed with another internalizingAb, hLL2, (humanized anti-CD22 Ab). These results demonstrated that thecytotoxicity effect of hLL1 on lymphoma cell lines is specificallydependent on crosslinking of the Ab on cell surface.

1. A method for treating a cancer that expresses CD74, comprisingadministering to a subject at least one naked humanized, human orchimeric anti-CD74 antibody or antigen-binding fragment thereof.
 2. Themethod of claim 1, wherein administering said naked anti-CD74 antibodyor fragment thereof is effective to treat the cancer.
 3. The method ofclaim 2, wherein administering said naked anti-CD74 antibody or fragmentthereof is effective to treat the cancer in the absence of any otheradministered antibody or fragment thereof.
 4. The method of claim 2,wherein administering said naked anti-CD74 antibody or fragment thereofis effective to treat the cancer in the absence of any otheradministered therapeutic agent.
 5. The method of claim 1, wherein theanti-CD74 antibody or fragment thereof is rapidly internalized.
 6. Themethod of claim 5, wherein 8×10⁶ antibody molecules per cell per day areinternalized.
 7. The method of claim 5, wherein 70% of anti-CD74antibody bound to the surface of Raji cells is internalized andcatabolized within three hours.
 8. The method of claim 1, wherein saidanti-CD74 antibody or fragment thereof induces apoptosis of cancercells.
 9. The method of claim 1, wherein the naked anti-CD74 antibody orfragment thereof is administered before, during or after theadministration of at least one therapeutic agent.
 10. The method ofclaim 9, wherein the therapeutic agent is selected from the groupconsisting of a second antibody, a second antibody fragment, animmunoconjugate, a fusion protein, a drug, a toxin, an antisenseoligonucleotide, a boron compound, an immunomodulator, a hormone, acytotoxic agent, an enzyme, an RNase, a recombinant RNase and aradionuclide.
 11. The method of claim 10, wherein said second antibody,second antibody fragment or immunoconjugate is reactive with a B-cell orT-cell antigen other than CD74.
 12. The method of claim 11, wherein saidantigen is selected from the group consisting of CD4, CD5, CD8, CD14,CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40,CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, Ia, HM1.24, tenascin andmature HLA-DR dimer.
 13. The method of claim 12, wherein said antigen isCD19, CD20 or CD22.
 14. The method of claim 10, wherein the drug isselected from the group consisting of a vinca alkaloid, ananthracycline, an epipodophyllotoxin, a taxane, an antimetabolite, analkylating agent, an antibiotic, a COX-2 inhibitor, an antimitoticagent, an antiangiogenic agent, an apoptotic agent, a camptothecan, anitrogen mustard, an alkyl sulfonate, a nitrosourea, a triazene, a folicacid analog, a pyrimidine analog, a purine analog and a platinumcoordination complex.
 15. The method of claim 10, wherein the drug isselected from the group consisting of doxorubicin, methotrexate, taxol,CPT-11, cyclophosphamide, etoposide, carmustine, vincristine,procarbazine, prednisone, bleomycin, leucovorin, phenyl butyrate,bryostatin-1 and dexamethasone.
 16. The method of claim 10, wherein thetoxin is selected from the group consisting of ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin andPseudomonas endotoxin.
 17. The method of claim 10, wherein theimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor, an interferon (IFN), an interleukin (IL) a tumornecrosis factor (TNF).
 18. The method of claim 17, wherein theimmunomodulator is selected from the group consisting of IFN-α, IFN-β,IFN-γ, erythropoietin, thrombopoietin, IL-1, IL-2, IL-3, IL-6, IL-10,IL-12, IL-15, IL-18, IL-21, TNF-α, TNF-β, granulocyte-colony stimulatingfactor (G-CSF), granulocyte macrophage-colony stimulating factor(GM-CSF) and the stem cell growth factor designated “S1 factor”.
 19. Themethod of claim 6, wherein the radionuclide is selected from the groupconsisting of ²²⁵Ac, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In, ¹³¹I, ¹²⁵I, ¹²³I,^(99m)Tc, ^(94m)Tc, ¹⁸⁶Re, ¹⁸⁸re, ¹⁷⁷Lu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi,³²P, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br and ²¹¹At.
 20. The method of claim 1, whereinsaid cancer is selected from the group consisting of a solid tumor,non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, multiple myeloma,a B-cell malignancy and a T-cell malignancy.
 21. The method of claim 20,wherein said solid tumor is selected from the group consisting ofmelanoma, carcinoma and sarcoma.
 22. The method of claim 21, whereinsaid solid tumor is selected from the group consisting of renal cancer,lung cancer, breast cancer, bladder cancer, pancreatic cancer, prostatecancer, intestinal cancer, stomach cancer, gastrointestinal cancer,melanoma and glioma.
 23. The method of claim 20, wherein said B-cellmalignancy is selected from the group consisting of non-Hodgkin'slymphoma, Hodgkin's lymphoma, indolent forms of B-cell lymphomas,aggressive forms of B-cell lymphomas, B-cell leukemias, chroniclymphocytic leukemia, acute lymphocytic leukemia, dendritic cellleukemia and multiple myeloma.
 24. The method of claim 1, wherein saidnaked anti-CD74 antibody or fragment thereof is administeredintravenously, intramuscularly, subcutaneously or parenterally.
 25. Themethod of claim 1, wherein said naked anti-CD74 antibody or fragmentthereof is a chimeric or humanized antibody or fragment thereofcomprising the light chain variable region complementarity-determiningregion (CDR) sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:19), CDR2(TVSNRFS; SEQ ID NO:20), and CDR3 (SQSSHVPPT; SEQ ID NO:21) and theheavy chain variable region CDR sequences CDR1 (NYGVN; SEQ ID NO:22),CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:23), and CDR3 (SRGKNEAWFAY; SEQ IDNO:24).
 26. The method of claim 1, wherein said naked anti-CD74 antibodyor fragment thereof competes for binding to CD74 with a monoclonal LL1antibody comprising the light chain variable regioncomplementarity-determining region (CDR) sequences CDR1(RSSQSLVHRNGNTYLH; SEQ ID NO:19), CDR2 (TVSNRFS; SEQ ID NO:20), and CDR3(SQSSHVPPT; SEQ ID NO:21) and the heavy chain variable region CDRsequences CDR1 (NYGVN; SEQ ID NO:22), CDR2 (WINPNTGEPTFDDDFKG; SEQ IDNO:23), and CDR3 (SRGKNEAWFAY; SEQ ID NO:24).
 27. A method for treatinga cancer that expresses CD74, comprising administering to a subject atleast one first immunoconjugate comprising a humanized, human orchimeric anti-CD74 antibody or antigen-binding fragment thereofconjugated to a therapeutic agent, wherein the therapeutic agent isselected from the group consisting of a second antibody, a secondantibody fragment, a second immunoconjugate, a fusion protein, a drug,an antisense oligonucleotide, a boron compound, an immunomodulator, ahormone and an enzyme.
 28. The method of claim 27, wherein said secondantibody, second antibody fragment or second immunoconjugate is reactivewith a B-cell or T-cell antigen other than CD74.
 29. The method of claim28, wherein said antigen is selected from the group consisting of CD4,CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33,CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, B7, MUC1, Ia,HM1.24, tenascin and mature HLA-DR dimer.
 30. The method of claim 29,wherein said antigen is CD19, CD20 or CD22.
 31. The method of claim 27,wherein the drug is selected from the group consisting of a vincaalkaloid, an anthracycline, an epipodophyllotoxin, a taxane, anantimetabolite, an alkylating agent, an antibiotic, a COX-2 inhibitor,an antimitotic agent, an antiangiogenic agent, an apoptotic agent, acamptothecan, a nitrogen mustard, an alkyl sulfonate, a nitrosourea, atriazene, a folic acid analog, a pyrimidine analog, a purine analog anda platinum coordination complex.
 32. The method of claim 27, wherein thedrug is selected from the group consisting of doxorubicin, methotrexate,taxol, CPT-11, cyclophosphamide, etoposide, carmustine, vincristine,procarbazine, prednisone, bleomycin, leucovorin, phenyl butyrate,bryostatin-1 and dexamethasone.
 33. The method of claim 27, wherein theimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor, an interferon (IFN), an interleukin (IL) a tumornecrosis factor (TNF).
 34. The method of claim 27, wherein theimmunomodulator is selected from the group consisting of IFN-α, IFN-β,IFN-γ, erythropoietin, thrombopoietin, IL-1, IL-2, IL-3, IL-6, IL-10,IL-12, IL-15, IL-18, IL-21, TNF-α, TNF-β, granulocyte-colony stimulatingfactor (G-CSF), granulocyte macrophage-colony stimulating factor(GM-CSF) and the stem cell growth factor designated “S1 factor”.
 35. Themethod of claim 27, wherein the anti-CD74 antibody or fragment thereofis rapidly internalized.
 36. The method of claim 35, wherein 8×10⁶antibody molecules per cell per day are internalized.
 37. The method ofclaim 35, wherein 70% of anti-CD74 antibody bound to the surface of Rajicells is internalized and catabolized within three hours.
 38. The methodof claim 27, further comprising administering to the subject at leastone other therapeutic agent.
 39. The method of claim 27, wherein saidcancer is selected from the group consisting of a solid tumor,non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, multiple myeloma,a B-cell malignancy and a T-cell malignancy.
 40. The method of claim 39,wherein said solid tumor is selected from the group consisting ofmelanoma, carcinoma and sarcoma.
 41. The method of claim 40, whereinsaid solid tumor is selected from the group consisting of renal cancer,lung cancer, breast cancer, bladder cancer, pancreatic cancer, prostatecancer, intestinal cancer, stomach cancer, gastrointestinal cancer,melanoma and glioma.
 42. The method of claim 39, wherein said B-cellmalignancy is selected from the group consisting of non-Hodgkin'slymphoma, Hodgkin's lymphoma, indolent forms of B-cell lymphomas,aggressive forms of B-cell lymphomas, B-cell leukemias, chroniclymphocytic leukemia, acute lymphocytic leukemia, dendritic cellleukemia and multiple myeloma.
 43. The method of claim 27, wherein saidfirst immunoconjugate is administered intravenously, intramuscularly,subcutaneously or parenterally.
 44. The method of claim 27, wherein saidanti-CD74 antibody or fragment thereof is a chimeric or humanizedantibody or fragment thereof comprising the light chain variable regioncomplementarity-determining region (CDR) sequences CDR1(RSSQSLVHRNGNTYLH; SEQ ID NO:19), CDR2 (TVSNRFS; SEQ ID NO:20), and CDR3(SQSSHVPPT; SEQ ID NO:21) and the heavy chain variable region CDRsequences CDR1 (NYGVN; SEQ ID NO:22), CDR2 (WINPNTGEPTFDDDFKG; SEQ IDNO:23), and CDR3 (SRGKNEAWFAY; SEQ ID NO:24).
 45. The method of claim27, wherein said anti-CD74 antibody or fragment thereof competes forbinding to CD74 with a monoclonal LL1 antibody comprising the lightchain variable region complementarity-determining region (CDR) sequencesCDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:19), CDR2 (TVSNRFS; SEQ ID NO:20), andCDR3 (SQSSHVPPT; SEQ ID NO:21) and the heavy chain variable region CDRsequences CDR1 (NYGVN; SEQ ID NO:22), CDR2 (WINPNTGEPTFDDDFKG; SEQ IDNO:23), and CDR3 (SRGKNEAWFAY; SEQ ID NO:24).